Anti-Siglec-9 antibodies and methods of use thereof

ABSTRACT

The present disclosure is generally directed to compositions that include antibodies, e.g., monoclonal, chimeric, humanized antibodies, antibody fragments, etc., that specifically bind one or more epitopes within a Siglec-9 protein, e.g., human Siglec-9 or a mammalian Siglec-9, and use of such compositions in preventing, reducing risk, or treating an individual in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application ofPCT/US2016/059443, filed internationally on Oct. 28, 2016, which claimsthe benefit of U.S. Provisional Application No. 62/248,231, filed Oct.29, 2015, each of which is hereby incorporated by reference in itsentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 735022001000SEQLIST.TXT,date recorded: Apr. 20, 2018, size: 197 KB).

FIELD OF THE INVENTION

This present disclosure relates to anti-Siglec-9 antibodies andtherapeutic uses of such antibodies.

BACKGROUND OF THE INVENTION

Sialic acid-binding Ig-like lectin-9 (Siglec-9), is a type 1,immunoglobulin-like, transmembrane protein expressed on immune andhematopoietic cells, including immature and mature myeloid cells, suchas monocytes, macrophages, dendritic cells, neutrophils, and microglialcells, as well as lymphoid cells, such as natural killer cells, andsubsets of T cells (Crocker et al. (2007) Nat Rev Immunol. 7:255-266;O'Reilly and Paulson (2009) Trends in Pharm. Sci. 30:5:240-248; andMacauley et al. (2014) Nat. Rev. 1 mm. 14: 653-666). Siglec-9 is amember of the Siglec family of lectins that bind sialic acid residues ofglycoproteins and glycolipids. One potential binding target for Siglecproteins is gangliosides; that is, glycolipids that consist of aceramide linked to a sialylated glycan. Most gangliosides share a commonlacto-ceramide core and one or more sialic acid residues. Diversity inthe Siglec ligands is generated by the addition of other neutral sugarsand sialic acid in different linkages, either branched or terminal, andmodification of sialic acid itself.

Fourteen Siglec proteins have been identified in humans and nine in micethat are comprised of 2-17 extracellular Ig domains including anamino-terminal V-set domain that contains the sialic acid-binding site.The sialic acid-binding region is located on the V-set Ig-like domain,which contains a two aromatic residues and one arginine motif highlyconserved in all Siglecs (Crocker et al. (2007) Nat Rev Immunol.7:255-266; McMillan and Crocker (2008) Carbohydr Res. 343:2050-2056; VonGunten and Bochner (2008) Ann NY Acad Sci. 1143:61-82; May et al. (1998)Mol Cell. 1:719-728; Crocker et al. (1999) Biochem J. 341:355-361; andCrocker and Varki (2001) Trends Immunol. 2:337-342). The binding sitesto sialylated ligands have been mapped by crystal structures with andwithout ligand bound (Attrill et al., (2006) J. Biol. Chem. 28132774-32783; Alphey et al. (2003) J. Biol. Chem. 278:5 3372-3377; Varkiet al., Glycobiology, 16 pp. 1R-27R; and May et al. (1998) Mol. Cell1:5:719-728). Since cell membranes are rich in sialic acids, ligandbinding by Siglecs can occur in cis and in trans, both affecting theirfunctional properties. Each Siglec has a distinct preference for bindingthe diverse types of sialylated glycans that are found on the surface ofmammalian cells (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; andCrocker et al. (2007) Nat Rev Immunol. 7:255-266). Most Siglec proteins,including Siglec-9, contain one or more immunoreceptor tyrosine-basedinhibitory motif (ITIM) sequences in their cytoplasmic tails, whichenable them as inhibitory receptors and negative regulators of immunefunctions through recruitment of the tyrosine phosphatases SHP1 and SHP2(Crocker et al. (2007) Nat Rev Immunol. 7:255-266; McMillan and Crocker(2008) Carbohydr Res. 343:2050-2056; and Von Gunten and Bochner (2008)Ann NY Acad Sci. 1143:61-82). Certain Siglecs contain immunoreceptortyrosine-based activating motif (ITAM) sequences in their cytoplasmictails, which enable them to act as activating receptors and positiveregulators of immune function through predicted recruitment of spleentyrosine kinase (Syk) (Macauley S M. et al., (2014) Nature ReviewsImmunology 14, 653-666). The Siglec protein family is associated withmultiple human diseases including, autoimmunity, susceptibility toinfection, multiple types of cancer including lymphoma, leukemia andacute myeloid leukemia, systemic lupus erythematosus, rheumatoidarthritis, neurodegenerative disorders, asthma, allergy, sepsis, chronicobstructive pulmonary disease, graft-versus-host disease, eosinophilia,and osteoporosis (Macauley S M. et al., (2014) Nature Reviews Immunology14, 653-666).

Siglec-9 was cloned in 2000 from peripheral blood mononuclear cells(Angata and Varki (2000) J. Biol. Chem. 275:29: 22127-22135) andselective expression was detected on granulocytes and monocytes. Anindependent group isolated Siglec-9 from HL-60 (human promyelocyticleukemia) cells and demonstrated expression on monocytes, neutrophils,NK cells and a small subset of T cells (Zhang et al. (2000) J. Biol.Chem. 275:29 22121-22126).

Siglec-9 contains an extracellular N-terminal Ig-like(immunoglobulin-like) V-type domain, two Ig-like C2-set domains as wellas two consensus ITIM motifs in its cytoplasmic domain. Expression ofSiglec-9 in COS cells demonstrated sialic acid-dependent binding of redblood cells, which is mediated by terminal α2-3 or α2-6 sialic acidlinkages (Angata and Varki (2000) J. Biol. Chem. 275: 22127-22135, Zhanget al. (2000) J. Biol. Chem. 275:29 22121-22126). It was furtherconfirmed that Siglec-9 is masked by endogenous cellular sialic acidsand binds to exogenous terminal α2-3 or α2-6 sialic acid probes onlyupon sialidase treatment of the cells (Yamaji (2002) J. Biol. Chem.277:8 6324-6332). Ligand specificity within the N-terminal V-set Ig-likedomain of Siglec-9 was mapped to a small region, Asn⁷⁰-Lys⁷⁵, byswapping Siglec-7 with Siglec-9 regions and vice versa. Acquisition ofthe respective Siglec ligand specificity within these amino acidresidues supports the notion that ligand specificity is dictated byinteractions in the variable C—C′ loop (Yamaji (2002) J. Biol. Chem.277:8 6324-6332). Pathogens have apparently subverted the sialic acid as“self” system as it has been reported that group B Streptococcus canbind Siglec-9 on human neutrophils thereby reducing the immune responseto the bacteria, which can either be pathogenic or commensal (Carlin etal (2009) Blood 113: 3333-3336). Other sources of in vivo Siglec-9sialic acid ligands are tumor-secreted mucins, such as MUC1, MUC2,MUC16; Siglec-9 was shown to bind mucins from the sera of cancerpatients (Ohta et al. (2010) Biochem. and Biophys. Res. Comm. 402:663-669; Belisle et al. (2010) Mol. Cancer 9:118).

Siglec-9 undergoes phosphorylation of Tyr-433, and Tyr-456 by tyrosinekinases, likely c-Src or Lck, and functions as an inhibitory receptor(Avril et al., (2004) J. Imm. 173: 6841-6849). Following phosphorylationon the proximal Tyr-433 in the ITIM domain, Siglec-9 binds SHP-2/PTPN11and SHP-1/PTPN6. The membrane distal ITIM motif does not appear tocontribute significantly as mutation did not preclude tyrosinephosphorylation or inhibitory function of Siglec-9. Siglec-9 was shownto inhibit FcεRI-mediated activities in rat basophilic leukemia cells,which have been previously used to characterize an inhibitory receptorclass expressed on NK cells called KIRs (Killer Ig-like receptors)(Avril et al., (2004) J. Imm. 173: 6841-6849). Phosphatase activity isadditionally associated with decreased intracellular calciummobilization, and decreased tyrosine phosphorylation on multipleproteins (Ulyanova, T., et al., (1999) Eur J Immunol 29, 3440-3449;Paul, S. P., et al., (2000). Blood 96, 483-490) as well as with blockadeof signal transduction and immune response, in part, throughdephosphorylation of signaling molecules on adjacent activatingreceptors, including those that contain ITAM motifs, pattern recognitionreceptors, Toll-like receptors and damage-associated molecular pattern(DAMP) receptors. It has been proposed that the association betweenITIM-containing Siglec receptors and activating receptors may bemediated by extracellular ligands that bind and bridge these receptors(Macauley S M. et al., (2014) Nature Reviews Immunology 14, 653-666).

Some, but not all, Siglec ligands induce receptor downregulation(Macauley S M. et al., (2014) Nature Reviews Immunology 14, 653-666).Ligand-induced receptor degradation has been reported for tyrosinekinase receptors (Monsonego-Oran et al., (2002) Febs letters 528, 83-89;and Fasen et al., (2008) Cell & Molecular Biology 9. 251-266), as wellas for steroid receptors (Callige et al., (2005) Mol. Cell. Biol. 25.4349-4358; and Pollenz et al., (2006) Chemico-Biological Interactions.164. 49-59). Siglec-9 is thought to be to constitutively recycled inacute myeloid leukemia (AML) cells and has been shown to mediate rapidendocytosis of an anti-Siglec-9 monoclonal antibody on these cells(Biedermann et al. (2007) Leuk. Res. 31:2:211-220). However, no decreasein cellular levels of Siglec-9 has been reported in either AML or normalprimary immune cells. Likewise, no receptor recycling orantibody-dependent receptor down regulation has been reported in anytype of primary cells. Expression of Siglec-9 on the cell surface isdependent in part on the membrane proximal ITIM motif, but not thedistal motif, according to mutational analysis performed in anoverexpression system (Biedermann et al. (2007) Leuk. Res.31:2:211-220).

Siglec-9 has been described as having immunomodulatory effects oncytokine production. Overexpression of Siglec-9 in a macrophage cellline and concomitant TLR stimulation has been shown to be associatedwith a decrease in production of proinflammatory cytokines TNF-alpha andIL-6, as well as upregulation of IL-10 (Ando et al. (2008) Biochem. AndBiophys. Res. Comm. 369:878-883). It has also been shown thattumor-produced mucins bind to Siglec-9, as well as immature DCs (Ohta etal. (2010) Biochem. and Biophys. Res. Comm. 402: 663-669). In thepresence of LPS and mucins, immature DCs produced less IL-12, but IL-10production was maintained. This suggests that Siglec-9 skews cytokineproduction from pro-inflammatory to anti-inflammatory, therebymaintaining an immunological state of tolerance as opposed to clearanceof offending pathogens, cancer, or other pathologies.

The inhibitory role of Siglec-9 has been further characterized in thefunction of natural killer cells and regulation of lymphoid cells, suchas T cells and neutrophils (Crocker et al., (2012) Ann. N Y Acad. Sci.1253, 102-111; Pillai et al., (2012) Annu. Rev. Immunol. 30, 357-392;von Gunten and Bochner (2008) Ann. N Y Acad. Sci. 1143, 61-82; Jandus etal. (2014) J. Clin. Invest. 124(4) 1810-1820; Ikehara et al. (2004) J.Biol. Chem. 279:41 43117-43125; and von Gunten et al. (2005) Blood106(4) 1423-1431). Functional studies in natural killer cells havedemonstrated that tumor cells expressing Siglec-9 binding sialic acidligands inhibit NK cell activation and tumor cell killing. Many humantumors robustly upregulate sialic acid ligands that bind Siglec-9, whichenables immune evasion and cancer progression (Jandus et al. (2014) J.Clinic. Invest. 124:4: 1810-1820). It is thought that sialic acidupregulation on tumors facilitates a state of “super self” that stronglyinhibits natural killer cell immunosurveillance (Macauley and Paulson(2014) Nat. Chem. Biol. 10:1: 7-8). In lymphoid lineage cells, Siglec-9has been shown to negatively regulate T cell receptor signaling via ITIMtyrosine phosphorylation and SHP-1 binding. Downstream TCR signalingmolecules ZAP-70 showed reduced phosphorylation on Tyr³¹⁹ and decreasedNFAT transcriptional activity. The inhibitory effects of Siglec-9 on TCRsignaling were reduced upon mutation of a conserved Arg residue in thesialic acid ligand-binding domain (Ikehara et al. (2004) J. Biol. Chem.279:41 43117-43125). In neutrophils, Siglec-9 engagement mediates celldeath via apoptotic and non-apoptotic mechanisms. Neutrophils derivedfrom non-diseased or rheumatoid arthritis and acute septic shockpatients underwent Siglec-9 dependent death, demonstrated by antibodycrosslinking. Septic or RA-patient-derived neutrophils demonstratedsignificantly more cell death upon Siglec-9 ligation; this increasecould be mimicked by short term pre-incubation with pro-inflammatorycytokines, suggesting that inflammation leads to priming of the Siglec-9death pathway (Belisle et al. (2010) Mol. Cancer 9:118).

The murine homolog of Siglec-9 is Siglec-E, which is 53% similar.Siglec-E was shown to bind human red blood cells in a sialic aciddependent manner, and functionally like Siglec-9, recruits SHP-1 andSHP-2 via ITIMs to mediate inhibitory signaling in immune cells (Yu etat Biochem. J. (2001) 353, 483-492). In mice, genetic inactivation ofSiglec-E does not lead to obvious developmental, histological, orbehavioral abnormalities; and Siglec-E-deficient mice breed normally,indicating that Siglec-E is not an essential gene and that its functionmay be limited to innate immunity (McMillan et al. (2013) Blood 121:11:2084-2094). Upon challenge of Siglec-E deficient mice with aerosol LPS,increased neutrophil recruitment in the lung was demonstrated, whichcould be reversed by blockade of the 32-integrin CD11b. The Siglec-Edeficient neutrophils were shown to have increased phosphorylation ofSyk and p38 MAPK in a CD11b-dependent manner. This suggests thatSiglec-E functions to suppress neutrophil recruitment in a model ofacute lung inflammation (McMillan et al. (2013) Blood 121:11:2084-2094). In a syngeneic cancer model, neutrophils from Siglec-Edeficient mice enhanced tumor cell killing ex vivo and demonstratedincreased ROS production and apoptosis inducing ligands such as TRAILand FasL (Laubli et al (2014) PNAS 111 (39) 14211-14216).

In oncology, Siglec-9 has been suggested as a therapeutic target foracute myeloid leukemia as it is expressed on primary AML cells, yetabsent from progenitors on numerous patient bone marrow samples(Biedermann et al. (2007) Leuk. Res. 31:2:211-220). In solid cancers,epithelial tumor cells produce heavily glycosylated mucins that bindSiglec-9, suggesting that blocking the increased ligand interactionswould be therapeutically beneficial (Ohta et al. (2010) Biochem. andBiophys. Res. Comm. 402: 663-669; Belisle et al. (2010) Mol. Cancer9:118). Furthermore, robust expression of Siglec-9 ligands and tumorinfiltrating Siglec-9⁺ immune cells were found in histological sectionsof colorectal, breast, ovarian, non-small lung cell, and prostate cancer(Laubli et al (2014) PNAS 111 (39) 14211-14216). A naturally occurringSiglec-9 K131Q (A391C) polymorphism (rs16988910) that reduces sialylligand binding was found to significantly improve early survival (<2years) in non-small-cell lung cancer patients, though the effect waslost after 2 years (Laubli et al (2014) PNAS 111 (39) 14211-14216).

It has recently been proposed that sialylglycoproteins expressed oncancer cells transduce ‘activation’ signals into tumor cells viaSiglec-9 binding, resulting in degradation of Focal adhesion kinase(FAK) and increased cell motility and invasion (Sabit et al. (2013) J.Biol. Chem. 288(49): 35417-35427). These results suggest thatSiglec-9-sialyl ligand interactions not only contribute to inhibitoryeffects on numerous cell types of the immune system, but could alsoenhance tumor metastasis via direct effects on cancer cells.

Antibodies to Siglec-9 have been described in, for example,WO2007049044, U.S. Pat. No. 8,394,382, EP1954318, and US20130302317.However, no antibodies that decrease the cellular levels of Siglec-9 orthat disrupt the interactions between Siglec-9 and one or more of itsligands have been reported.

Accordingly, there is a need for therapeutic antibodies thatspecifically bind Siglec-9 and reduce Siglec-9 expression on the cellsurface, reduce interactions between Siglec-9 and one or more Siglec-9ligands, and/or reduce one or more Siglec-9 activities in order to treatone or more diseases, disorders, and conditions associated withundesired Siglec-9 activity.

All references cited herein, including patents, patent applications andpublications, are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present disclosure is generally directed to Siglec-9 agents, such asanti-Siglec-9 antibodies, and methods of using such Siglec-9 agents. Themethods provided herein find use in preventing, reducing risk, ortreating an individual having dementia, frontotemporal dementia,Alzheimer's disease, vascular dementia, mixed dementia,Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophiclateral sclerosis, Huntington's disease, taupathy disease, Nasu-Hakoladisease, stroke, acute trauma, chronic trauma, lupus, acute and chroniccolitis, rheumatoid arthritis, wound healing, Crohn's disease,inflammatory bowel disease, ulcerative colitis, obesity, malaria,essential tremor, central nervous system lupus, Behcet's disease,Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,Shy-Drager syndrome, progressive supranuclear palsy, cortical basalganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, solid andblood cancer, bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9and/or Siglec-9 ligands, thyroid cancer, infections, CNS herpes,parasitic infections, Trypanosome infection, Cruzi infection,Pseudomonas aeruginosa infection, Leishmania donovani infection, group BStreptococcus infection, Campylobacter jejuni infection, Neisseriameningiditis infection, type I HIV, and Haemophilus influenza. Themethods provided herein also find use in inducing or promoting thesurvival, maturation, functionality, migration, or proliferation of oneor more immune cells in an individual in need thereof. The methodsprovided herein find further use in decreasing the activity,functionality, or survival of regulatory T cells, tumor-imbeddedimmununosuppressor dendritic cells, tumor-imbedded immunosuppressormacrophages, neutrophils, natural killer (NK) cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, neutrophils, NK cells,acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL)cell, or chronic myeloid leukemia (CML) cell in an individual in needthereof. The methods provided herein also find use in decreasingcellular levels of Siglec-9.

Certain aspects of the present disclosure are based, at least in part,on the identification of anti-Siglec-9 antibodies that are capable ofdecreasing cell surface levels of Siglec-9 on human primary immune cellsand Siglec-9-expressing cell lines and/or that are capable of inhibitingthe binding of Siglec-9 ligands to Siglec-9 (see, e.g., Examples 3-5).

Accordingly, certain aspects of the present disclosure relate to anisolated (e.g., monoclonal) anti-Siglec-9 antibody, wherein theanti-Siglec-9 antibody decreases cellular levels of Siglec-9. In someembodiments, the anti-Siglec-9 antibody decreases cellular levels ofSiglec-9 without inhibiting interaction between Siglec-9 and one or moreSiglec-9 ligands. In some embodiments, the antibody further inhibitsinteraction between Siglec-9 and one or more Siglec-9 ligands. Otheraspects of the present disclosure relate to an isolated (e.g.,monoclonal) anti-Siglec-9 antibody, wherein the anti-Siglec-9 antibodydecreases cellular levels of Siglec-9 and inhibits interaction betweenSiglec-9 and one or more Siglec-9 ligands.

In some embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody decreases cell surface levels ofSiglec-9, decreases intracellular levels of Siglec-9, decreases totallevels of Siglec-9, or any combination thereof. In some embodiments thatmay be combined with any of the preceding embodiments, the anti-Siglec-9antibody induces Siglec-9 degradation, Siglec-9 cleavage, Siglec-9internalization, Siglec-9 shedding, downregulation of Siglec-9expression, or any combination thereof. In some embodiments that may becombined with any of the preceding embodiments, the antibody decreasescellular levels of Siglec-9 in vivo. In some embodiments that may becombined with any of the preceding embodiments, the anti-Siglec-9antibody inhibits cell surface clustering of Siglec-9. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody inhibits one or more Siglec-9 activities. Insome embodiments that may be combined with any of the precedingembodiments, the one or more Siglec-9 activities selected from the groupconsisting of: (a) Siglec-9 binding to one or more Siglec-9 ligands,optionally wherein the one or more Siglec-9 ligands are selected fromthe group consisting of sialic acid-containing glycoproteins, sialicacid-containing glycolipids, and any combination thereof; (b) Siglec-9binding to SHP1 or SHP2; (c) phosphorylation of Tyr-433, Tyr-456, orboth, induced by one or more SRC family tyrosine kinases, optionally,wherein the one or more SRC family tyrosine kinases are selected fromthe group consisting of Syk, LCK, FYM, and ZAP-70; (d) modulatedexpression of one or more pro-inflammatory cytokines, optionally whereinthe one or more pro-inflammatory cytokines are selected from a groupconsisting FN-α4, IFN-beta, IL-1β, IL-1alpha, TNF-α, IL-6, IL-8, CRP,IL-20 family members, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12,IL-17, IL-18, IL-33, MCP-1, and MIP-1-beta; (e) modulated expression ofone or more pro-inflammatory cytokines in one or more cells selectedfrom the group consisting of macrophages, neutrophils, NK cells,dendritic cells, bone marrow-derived dendritic cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,and microglial cells; (f) modulated expression of one or moreanti-inflammatory cytokines, optionally wherein the one or moreanti-inflammatory cytokines are selected from the group consisting ofIL-4, IL-10, IL-13, IL-35, IL-16, TGF-beta, IL-1Ra, G-CSF, and solublereceptors for TNF, IFN-beta1a, IFN-beta1b, or IL-6; (g) modulatedexpression of one or more anti-inflammatory cytokines in one or morecells selected from the group consisting of macrophages, neutrophils, NKcells, dendritic cells, bone marrow-derived dendritic cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,and microglial cells; (h) modulate expression of one or more proteinsselected from the group consisting of C1qa, C1qB, C1qC, C1s, C1R, C4,C2, C3, ITGB2, HMOX1, LAT2, CASP1, CSTA, VSIG4, MS4A4A, C3AR1, GPX1,TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX, and PYCARD; (i) inhibitionof extracellular signal-regulated kinase (ERK) phosphorylation; (j)decreasing tyrosine phosphorylation on one or more cellular proteins,optionally, wherein the one or more cellular proteins comprise ZAP-70and the tyrosine phosphorylation occurs on Tyr-319 of ZAP-70; (k)modulated expression of C—C chemokine receptor 7 (CCR7); (l) inhibitionof microglial cell chemotaxis toward CCL19-expressing andCCL21-expressing cells; (m) decreasing T cell proliferation induced byone or more cells selected from the group consisting of dendritic cells,bone marrow-derived dendritic cells, monocytes, microglia, M1 microglia,activated M1 microglia, M2 microglia, macrophages, neutrophils, NKcells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1macrophages, activated M1 neutrophils, activated M1 NK cells, M2macrophages, M2 neutrophils, and M2 NK cells; (n) inhibition ofosteoclast production, decreased rate of osteoclastogenesis, or both;(o) decreasing survival of one or more cells selected from the groupconsisting of dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; (p) decreasing proliferation of one or more cells selectedfrom the group consisting of dendritic cells, bone marrow-deriveddendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; (q) inhibiting migration of one or morecells selected from the group consisting of dendritic cells, bonemarrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages,activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helpercells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1microglia, activated M1 microglia, and M2 microglia; (r) inhibiting oneor more functions of one or more cells selected from the groupconsisting of dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; (s) inhibiting maturation of one or more cells selected fromthe group consisting of dendritic cells, bone marrow-derived dendriticcells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; (t) inhibition of one or more types ofclearance selected from the group consisting of apoptotic neuronclearance, nerve tissue debris clearance, dysfunctional synapseclearance, non-nerve tissue debris clearance, bacteria clearance, otherforeign body clearance, disease-causing protein clearance,disease-causing peptide clearance, and tumor cell clearance; optionallywherein the disease-causing protein is selected from the groupconsisting of amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides and the tumor cell is from a cancer selected fromthe group consisting of bladder cancer, brain cancer, breast cancer,colon cancer, rectal cancer, endometrial cancer, kidney cancer, renalcell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma,non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovariancancer, fibrosarcoma, and thyroid cancer; (u) inhibition of phagocytosisof one or more of apoptotic neurons, nerve tissue debris, dysfunctionalsynapses, non-nerve tissue debris, bacteria, other foreign bodies,disease-causing proteins, disease-causing peptides, disease-causingnucleic acids, or tumor cells; optionally wherein the disease-causingnucleic acids are antisense GGCCCC (G2C4) repeat-expansion RNA, thedisease-causing proteins are selected from the group consisting ofamyloid beta, oligomeric amyloid beta, amyloid beta plaques, amyloidprecursor protein or fragments thereof, Tau, IAPP, alpha-synuclein,TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RANprotein, prion protein, PrPSc, huntingtin, calcitonin, superoxidedismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8,ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides, and the tumor cells are from a cancer selectedfrom the group consisting of bladder cancer, brain cancer, breastcancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer,renal cell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma,non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovariancancer, fibrosarcoma, or thyroid cancer; (v) binding to Siglec-9 ligandon tumor cells; (w) binding to Siglec-9 ligand on cells selected fromthe group consisting of neutrophils, dendritic cells, bonemarrow-derived dendritic cells, monocytes, microglia, macrophages, andNK cells; (x) inhibition of tumor cell killing by one or more ofmicroglia, macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, neutrophils, T cells, T helper cells, orcytotoxic T cells; (y) inhibiting anti-tumor cell proliferation activityof one or more of microglia, macrophages, neutrophils, NK cells,dendritic cells, bone marrow-derived dendritic cells, neutrophils, Tcells, T helper cells, or cytotoxic T cells; (z) inhibition ofanti-tumor cell metastasis activity of one or more of microglia,macrophages, neutrophils, NK cells, dendritic cells, bone marrow-deriveddendritic cells, neutrophils, T cells, T helper cells, or cytotoxic Tcells; (aa) inhibition of one or more ITAM motif containing receptors,optionally wherein the one or more ITAM motif containing receptors areselected from the group consisting of TREM1, TREM2, Sirp beta, FcgR,DAP10, and DAP12; (bb) inhibition of signaling by one or more patternrecognition receptors (PRRs), optionally wherein the one or more PRRsare selected from the group consisting of receptors that identifypathogen-associated molecular patterns (PAMPs), receptors that identifydamage-associated molecular patterns (DAMPs), and any combinationthereof; (cc) inhibition of one or more receptors comprising the motifD/Ex₀₋₂YxxL/IX₆₋₈YxxL/I (SEQ ID NO: 252); (dd) inhibition of signalingby one or more Toll-like receptors; (ee) inhibition of the JAK-STATsignaling pathway; (ff) inhibition of nuclear factorkappa-light-chain-enhancer of activated B cells (NFκB); (gg)de-phosphorylation of an ITAM motif containing receptor; (hh) modulatedexpression of one or more inflammatory receptors, proteins of thecomplement cascade, and/or receptors that are expressed on immune cells,optionally wherein the one or more inflammatory receptors, proteins ofthe complement cascade, and/or receptors that are expressed on immunecells comprise CD86, C1qa, C1qB, C1qC, C1s, C1R, C4, C2, C3, ITGB2,HMOX1, LAT2, CASP1, CSTA, VSIG4, MS4A4A, C3AR1, GPX1, TyroBP, ALOX5AP,ITGAM, SLC7A7, CD4, ITGAX, and/or PYCARD, and the one or moreinflammatory receptors, proteins of the complement cascade, and/orreceptors that are expressed on immune cells are expressed on one ormore of microglia, macrophages, neutrophils, NK cells, dendritic cells,bone marrow-derived dendritic cells, neutrophils, T cells, T helpercells, or cytotoxic T cells; (ii) increasing expression of one or moreSiglec-9-dependent genes; (jj) normalization of disruptedSiglec-9-dependent gene expression; (kk) decreasing expression of one ormore ITAM-dependent genes, optionally wherein the one moreITAM-dependent genes are activated by nuclear factor of activated Tcells (NFAT) transcription factors; (ll) promoting differentiation ofone or more of immunosuppressor dendritic cells, immunosuppressormacrophages, immunosuppressor neutrophils, immunosuppressor NK cells,myeloid derived suppressor cells, tumor-associated macrophages,tumor-associated neutrophils, tumor-associated NK cells, and regulatoryT cells; (mm) promoting or rescuing functionality of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, and regulatory T cells; (nn)increasing infiltration of one or more of immunosuppressor dendriticcells, immunosuppressor macrophages, immunosuppressor neutrophils,immunosuppressor NK cells, myeloid-derived suppressor cells,tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, and regulatory T cells into tumors; (oo)increasing the number of tumor-promoting myeloid/granulocyticimmune-suppressive cells in a tumor, in peripheral blood, or otherlymphoid organ; (pp) enhancing tumor-promoting activity ofmyeloid-derived suppressor cells; (qq) increasing expression oftumor-promoting cytokines in a tumor or in peripheral blood, optionallywherein the tumor-promoting cytokines are TGF-beta or IL-10; (rr)increasing tumor infiltration of tumor-promoting FoxP3+ regulatory Tlymphocytes; (ss) enhancing tumor-promoting activity of myeloid-derivedsuppressor cells (MDSC); (tt) decreasing activation of tumor-specific Tlymphocytes with tumor killing potential; (uu) decreasing infiltrationof tumor-specific NK cells with tumor killing potential; (vv) decreasingthe tumor killing potential of NK cells; (ww) decreasing infiltration oftumor-specific B lymphocytes with potential to enhance immune response;(xx) decreasing infiltration of tumor-specific T lymphocytes with tumorkilling potential; (yy) increasing tumor volume; (zz) increasing tumorgrowth rate; (aaa) increasing metastasis; (bbb) increasing rate of tumorrecurrence; (ccc) decreasing efficacy of one or more immune-therapiesthat modulate anti-tumor T cell responses, optionally wherein the one ormore immune-therapies are immune-therapies that target one or moretarget proteins selected from the group consisting of PD1/PDL1, CD40,OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1,B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG, DR-5, TREM1,TREM2, CSF-1 receptor, and any combination thereof, or of one or morecancer vaccines; (ddd) inhibition of PLCγ/PKC/calcium mobilization; and(eee) inhibition of PI3K/Akt, Ras/MAPK signaling. In some embodimentsthat may be combined with any of the preceding embodiments, the one ormore Siglec-9 activities are selected from the group consisting of: (a)enhancing infiltration of one or more of immunosuppressor dendriticcells, immunosuppressor macrophages, myeloid derived suppressor cells,tumor-associated macrophages, immunosuppressor neutrophils,non-tumorigenic CD45⁺CD14⁺ myeloid cells, and regulatory T cells intotumors; (b) increasing number of tumor-promoting myeloid/granulocyticimmune-suppressive cells in a tumor, in peripheral blood, or otherlymphoid organ; (r) enhancing tumor-promoting activity ofnon-tumorigenic myeloid-derived suppressor cells and/or non-tumorigenicCD45⁺CD14⁺ myeloid cells; (c) enhancing survival of non-tumorigenicmyeloid-derived suppressor cells (MDSC) and/or non-tumorigenicCD45⁺CD14⁺ myeloid cells; (d) decreasing activation of tumor-specific Tlymphocytes with tumor killing potential; (e) decreasing activation ofCD45⁺CD3⁺ T lymphocytes with tumor killing potential; (f) decreasinginfiltration of tumor-specific NK cells with tumor killing potential;(g) decreasing infiltration of tumor-specific B lymphocytes withpotential to enhance immune response; (h) decreasing infiltration oftumor-specific T lymphocytes with tumor killing potential; and (i)decreasing infiltration of CD45⁺CD3⁺ T lymphocytes. In some embodimentsthat may be combined with any of the preceding embodiments, the one ormore Siglec-9 activities selected from the group consisting of: (a)Siglec-9 binding to one or more Siglec-9 ligands, optionally wherein theone or more Siglec-9 ligands are selected from the group consisting ofsialic acid-containing glycoproteins, sialic acid-containingglycolipids, and any combination thereof; (b) decreasing proliferationof one or more cells selected from the group consisting of dendriticcells, hone marrow-derived dendritic cells, macrophages, neutrophils, NKcells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1macrophages, activated M1 neutrophils, activated M1 NK cells, M2macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, Tcells, T helper cells, cytotoxic T cells, granulocytes, neutrophils,microglia, M1 microglia, activated M1 microglia, and M2 microglia; (c)inhibiting migration of one or more cells selected from the groupconsisting of dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; (d) inhibiting one or more functions of one or more cellsselected from the group consisting of dendritic cells, bonemarrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages,activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helpercells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1microglia, activated M1 microglia, and M2 microglia; (e) inhibition ofone or more types of clearance selected from the group consisting ofapoptotic neuron clearance, nerve tissue debris clearance, dysfunctionalsynapse clearance, non-nerve tissue debris clearance, bacteriaclearance, other foreign body clearance, disease-causing proteinclearance, disease-causing peptide clearance, and tumor cell clearance;optionally wherein the disease-causing protein is selected from thegroup consisting of amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AT, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides and the tumor cell is from a cancer selected fromthe group consisting of bladder cancer, brain cancer, breast cancer,colon cancer, rectal cancer, endometrial cancer, kidney cancer, renalcell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma,non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovariancancer, fibrosarcoma, and thyroid cancer; (f) inhibition of tumor cellkilling by one or more of microglia, macrophages, neutrophils, NK cells,dendritic cells, bone marrow-derived dendritic cells, neutrophils, Tcells, T helper cells, or cytotoxic T cells; (g) inhibiting anti-tumorcell proliferation activity of one or more of microglia, macrophages,neutrophils, NK cells, dendritic cells, hone marrow-derived dendriticcells, neutrophils, T cells, T helper cells, or cytotoxic T cells; (h)modulating expression of one or more inflammatory receptors, optionallywherein the one or more inflammatory receptors comprise CD86 and the oneor more inflammatory receptors are expressed on one or more ofmicroglia, macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, neutrophils, T cells, T helper cells, orcytotoxic T cells; (i) promoting or rescuing functionality of one ormore of immunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, and regulatory T cells; (j)increasing infiltration of one or more of immunosuppressor dendriticcells, immunosuppressor macrophages, immunosuppressor neutrophils,immunosuppressor NK cells, myeloid-derived suppressor cells,tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, non-tumorigenic CD45⁺CD14⁺ myeloid cells, andregulatory T cells into tumors; (k) increasing the number oftumor-promoting myeloid/granulocytic immune-suppressive cells and/ornon-tumorigenic CD45⁺CD14⁺ myeloid cells in a tumor, in peripheralblood, or other lymphoid organ; (l) enhancing tumor-promoting activityof myeloid-derived suppressor cells and/or non-tumorigenic CD45⁺CD14⁺myeloid cells; (m) enhancing survival of non-tumorigenic myeloid-derivedsuppressor cells and/or non-tumorigenic CD45⁺CD14⁺ myeloid cells; (n)decreasing activation of tumor-specific T lymphocytes with tumor killingpotential; (o) decreasing infiltration of tumor-specific NK cells withtumor killing potential; (p) increasing tumor volume; (q) increasingtumor growth rate; and (r) decreasing efficacy of one or moreimmune-therapies that modulate anti-tumor T cell responses, optionallywherein the one or more immune-therapies are immune-therapies thattarget one or more target proteins selected from the group consisting ofPD1/PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4,PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR,GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39,CD73, CSF-1 receptor, and any combination thereof, or of one or morecancer vaccines. In some embodiments that may be combined with any ofthe preceding embodiments, the anti-Siglec-9 antibody exhibits one ormore activities selected from the group consisting of consisting of: (a)increasing the number of tumor infiltrating CD3⁺ T cells; (b) decreasingcellular levels of CD33 in non-tumorigenic CD14⁺ myeloid cells,optionally wherein the non-tumorigenic CD14⁺ myeloid cells are tumorinfiltrating cells or optionally wherein the non-tumorigenic CD14⁺myeloid cells are present in blood; (c) reducing the number ofnon-tumorigenic CD14⁺ myeloid cells, optionally wherein thenon-tumorigenic CD14⁺ myeloid cells are tumor infiltrating cells oroptionally wherein the non-tumorigenic CD14⁺ myeloid cells are presentin blood; (d) reducing PD-L1 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (e) reducing PD-L2 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (f) reducing B7-H2 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (g) reducingB7-H3 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (h)reducing CD200R levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (i) reducing CD163 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (j) reducing CD206 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (k) decreasing tumor growthrate of solid tumors; (l) reducing tumor volume; (m) increasing efficacyof one or more PD-1 inhibitors; (n) increasing efficacy of one or morecheckpoint inhibitor therapies and/or immune-modulating therapies,optionally wherein the one or more checkpoint inhibitor therapies and/orimmune-modulating therapies target one or more of CTLA4, the adenosinepathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; (o)increasing efficacy of one or more chemotherapy agents, optionallywherein the one or more of the chemotherapy agents are gemcitabine,capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin(Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®),5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin(Paraplatin®), and any combination thereof; (p) increasing proliferationof T cells in the presence of non-tumorigenic myeloid-derived suppressorcells (MDSC); (q) inhibiting differentiation, survival, and/or one ormore functions of non-tumorigenic myeloid-derived suppressor cells(MDSC); and (r) killing CD33-expressing immunosuppressor non-tumorigenicmyeloid cells and/or non-tumorigenic CD14-expressing cells in solidtumors and associated blood vessels when conjugated to a chemical orradioactive toxin. In some embodiments that may be combined with any ofthe preceding embodiments, the one or more Siglec-9 ligands are selectedfrom the group consisting of Siglec-9 ligands expressed on red bloodcells, Siglec-9 ligands expressed on bacterial cells, Siglec-9 ligandsexpressed on apoptotic cells, Siglec-9 ligands expressed on nerve cells,Siglec-9 ligands expressed on glia cells, Siglec-9 ligands expressed onmicroglia, Siglec-9 ligands expressed on astrocytes, Siglec-9 ligandsexpressed on tumor cells, Siglec-9 ligands expressed on viruses,Siglec-9 ligands expressed on dendritic cells, Siglec-9 ligands bound tobeta amyloid plaques, Siglec-9 ligands bound to Tau tangles, Siglec-9ligands on disease-causing proteins, Siglec-9 ligands on disease-causingpeptides, Siglec-9 ligands expressed on macrophages, Siglec-9 ligandsexpressed on neutrophils, Siglec-9 ligands expressed on natural killercells, Siglec-9 ligands expressed on monocytes, Siglec-9 ligandsexpressed on T cells, Siglec-9 ligands expressed on T helper cells,Siglec-9 ligands expressed on cytotoxic T cells, Siglec-9 ligandsexpressed on B cells, Siglec-9 ligands expressed on tumor-imbeddedimmunosuppressor dendritic cells, Siglec-9 ligands expressed ontumor-imbedded immunosuppressor macrophages, Siglec-9 ligands expressedon myeloid-derived suppressor cells, Siglec-9 ligands expressed onregulatory T cells, secreted mucins, sialic acid, sialic acid-containingglycolipids, sialic acid-containing glycoproteins, alpha-2,8-disialylcontaining glycolipids, branched alpha-2,6-linked sialic acid-containingglycoproteins, terminal alpha-2,6-linked sialic acid-containingglycolipids, terminal alpha-2,3-linked sialic acid-containingglycoproteins, and disialogangliosides. In some embodiments that may becombined with any of the preceding embodiments, the cellular levels ofSiglec-9 are measured on primary cells selected from the groupconsisting of dendritic cells, bone marrow-derived dendritic cells,monocytes, microglia, macrophages, neutrophils, and NK cells, or on celllines, and wherein the cellular levels of Siglec-9 are measuredutilizing an in vitro cell assay. In some embodiments that may becombined with any of the preceding embodiments, the anti-Siglec-9antibody does not reduce TREM2 expression. In some embodiments that maybe combined with any of the preceding embodiments, the anti-Siglec-9antibody exhibits one or more activities selected from the groupconsisting of consisting of: (a) increasing the number of tumorinfiltrating CD3⁺ T cells; (b) decreasing cellular levels of Siglec-9 innon-tumorigenic CD14+ myeloid cells, optionally wherein thenon-tumorigenic CD14+ myeloid cells are tumor infiltrating cells oroptionally wherein the non-tumorigenic CD14+ myeloid cells are presentin blood; (c) reducing the number of non-tumorigenic CD14+ myeloidcells, optionally wherein the non-tumorigenic CD14+ myeloid cells aretumor infiltrating cells or optionally wherein the non-tumorigenic CD14+myeloid cells are present in blood; (d) reducing PD-L1 levels in one ormore cells, optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (e) reducing PD-L2 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (f) reducingB7-H2 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (g)reducing B7-H3 levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (h) reducing CD200R levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (i) reducing CD163 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (j) reducing CD206 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (k) decreasingtumor growth rate of solid tumors; (l) reducing tumor volume; (m)increasing efficacy of one or more PD-1 inhibitors; (n) increasingefficacy of one or more checkpoint inhibitor therapies and/orimmune-modulating therapies, optionally wherein the one or morecheckpoint inhibitor therapies and/or immune-modulating therapies targetone or more of CTLA4, the adenosine pathway, PD-L1, PD-L2, PD-L1, PD-L2,OX40, TIM3, LAG3, or any combination thereof; (o) increasing efficacy ofone or more chemotherapy agents, optionally wherein the one or more ofthe chemotherapy agents are gemcitabine, capecitabine, anthracyclines,doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel(Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide(Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (p)increasing proliferation of T cells in the presence of non-tumorigenicmyeloid-derived suppressor cells (MDSC); (q) inhibiting differentiation,survival, and/or one or more functions of non-tumorigenicmyeloid-derived suppressor cells (MDSC); and (r) killingSiglec-9-expressing immunosuppressor non-tumorigenic myeloid cellsand/or non-tumorigenic CD14-expressing cells in solid tumors andassociated blood vessels when conjugated to a chemical or radioactivetoxin.

In some embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody binds a discontinuous Siglec-9epitope. In some embodiments that may be combined with any of thepreceding embodiments, the discontinuous Siglec-9 epitope comprises twoor more peptides, three or more peptides, four or more peptides, five ormore peptides, six or more peptides, seven or more peptides, eight ormore peptides, nine or more peptides, or 10 or more peptides. In someembodiments that may be combined with any of the preceding embodiments,each of the peptides comprise five or more, six or more, seven or more,eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 ormore 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 ormore, or 20 or more amino acid residues of the amino acid sequence ofSEQ ID NO: 1; or five or more, six or more, seven or more, eight ormore, nine or more, 10 or more, 11 or more, 12 or more, 13 or more 14 ormore, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20or more amino acid residues on a mammalian Siglec-9 proteincorresponding to the amino acid sequence of SEQ ID NO: 1. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody binds to a conformational epitope ofSiglec-9. In some embodiments that may be combined with any of thepreceding embodiments, the anti-Siglec-9 antibody binds to one or moreamino acids within amino acid residues 20-347, 20-140, 141-347, 146-347,146-229, 236-336, or 146-347 of SEQ ID NO: 1; or within amino acidresidues on a mammalian Siglec-9 protein corresponding to amino acidresidues 20-347, 20-140, 141-347, 146-347, 146-229, 236-336, or 146-347of SEQ ID NO: 1. In some embodiments that may be combined with any ofthe preceding embodiments, the anti-Siglec-9 antibody binds to one ormore amino acids within amino acid residues selected from the groupconsisting of: i. amino acid residues 62-76 of SEQ ID NO: 1, or aminoacid residues on a mammalian Siglec-9 protein corresponding to aminoacid residues 62-76 of SEQ ID NO: 1; ii. amino acid residues 62-76 and86-92 of SEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9protein corresponding to amino acid residues 62-76 and 86-92 of SEQ IDNO: 1; iii. amino acid residues 86-92 of SEQ ID NO: 1, or amino acidresidues on a mammalian Siglec-9 protein corresponding to amino acidresidues 86-92 of SEQ ID NO: 1; iv. amino acid residues 86-96 of SEQ IDNO: 1, or amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 86-96 of SEQ ID NO: 1; v. aminoacid residues 86-96 and 105-116 of SEQ ID NO: 1, or amino acid residueson a mammalian Siglec-9 protein corresponding to amino acid residues86-96 and 105-116 of SEQ ID NO: 1; vi. amino acid residues 105-116 ofSEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 105-116 of SEQ ID NO: 1; vii. aminoacid residues 107-115 of SEQ ID NO: 1, or amino acid residues on amammalian Siglec-9 protein corresponding to amino acid residues 107-115of SEQ ID NO: 1; and viii. amino acid residues 185-194 of SEQ ID NO: 1,or amino acid residues on a mammalian Siglec-9 protein corresponding toamino acid residues 185-194 of SEQ ID NO: 1. In some embodiments thatmay be combined with any of the preceding embodiments, the anti-Siglec-9antibody binds to one or more amino acids within amino acid residues62-76 of SEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9protein corresponding to amino acid residues 62-76 of SEQ ID NO: 1. Insome embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody binds to one or more amino acidswithin amino acid residues 62-76 and 86-92 of SEQ ID NO: 1, or aminoacid residues on a mammalian Siglec-9 protein corresponding to aminoacid residues 62-76 and 86-92 of SEQ ID NO: 1. In some embodiments thatmay be combined with any of the preceding embodiments, the anti-Siglec-9antibody binds to one or more amino acids within amino acid residues86-92 of SEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9protein corresponding to amino acid residues 86-92 of SEQ ID NO: 1. Insome embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody binds to one or more amino acidswithin amino acid residues 86-96 of SEQ ID NO: 1, or amino acid residueson a mammalian Siglec-9 protein corresponding to amino acid residues86-96 of SEQ ID NO: 1. In some embodiments that may be combined with anyof the preceding embodiments, the anti-Siglec-9 antibody binds to one ormore amino acids within amino acid residues 86-96 and 105-116 of SEQ IDNO: 1, or amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 86-96 and 105-116 of SEQ ID NO: 1.In some embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody binds to one or more amino acidswithin amino acid residues 105-116 of SEQ ID NO: 1, or amino acidresidues on a mammalian Siglec-9 protein corresponding to amino acidresidues 105-116 of SEQ ID NO: 1. In some embodiments that may becombined with any of the preceding embodiments, the anti-Siglec-9antibody binds to one or more amino acids within amino acid residues107-115 of SEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9protein corresponding to amino acid residues 107-115 of SEQ ID NO: 1. Insome embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody binds to one or more amino acidswithin amino acid residues 185-194 of SEQ ID NO: 1, or amino acidresidues on a mammalian Siglec-9 protein corresponding to amino acidresidues 185-194 of SEQ ID NO: 1. In some embodiments that may becombined with any of the preceding embodiments, the anti-Siglec-9antibody binds to one or more amino acid residues selected from thegroup consisting of L22, H48, W50, I51, Y52, K123, I126, D189, P190,R194 of SEQ ID NO: 1, or one or more amino acid residues on a mammalianSiglec-7 protein corresponding to an amino acid residue selected fromthe group consisting of L22, H48, W50, I51, Y52, K123, I126, D189, P190,R194 of SEQ ID NO: 1. In some embodiments that may be combined with anyof the preceding embodiments, the anti-Siglec-9 antibody competes withone or more antibodies selected from the group consisting of 2D4, 2D5,5B1, 6B2, 6D8, 7H12, 5C6, 12B12, 17C2, and any combination thereof forbinding to Siglec-9.

In some embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain, the heavy chain variable domain, or both comprise atleast one, two, three, four, five, or six HVRs selected from HVR-L1,HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 of a monoclonal antibodyselected from the group consisting of: 2D4, 2D5, 5B1, 6B2, 6D8, 7H12,5C6, 12B12, and 17C2. In some embodiments that may be combined with anyof the preceding embodiments: (a) the HVR-L1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 6-9, 172, and173; or (b) the HVR-L2 comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 10-13, 174, and 175; or (c) theHVR-L3 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14-18, 176, and 177; or (d) the HVR-H1comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 19-21, 178, and 179; or (e) the HVR-H2 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 22-25,180, and 181; or (f) the HVR-H3 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 26-29, 182, and 183.In some embodiments that may be combined with any of the precedingembodiments: (a) the HVR-L1 comprises the amino acid sequence of SEQ IDNO: 6, the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 10,the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 14, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 19, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 22, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 26; or (b) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 7, the HVR-L2 comprisesthe amino acid sequence of SEQ ID NO: 11, the HVR-L3 comprises the aminoacid sequence of SEQ ID NO: 15, the HVR-H1 comprises the amino acidsequence of SEQ ID NO: 20, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO: 23, and the HVR-H3 comprises the amino acid sequence ofSEQ ID NO: 27; or (c) the HVR-L1 comprises the amino acid sequence ofSEQ ID NO: 8, the HVR-L2 comprises the amino acid sequence of SEQ ID NO:12, the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 16, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 21, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 24, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 28; or (d) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 9, the HVR-L2 comprisesthe amino acid sequence of SEQ ID NO: 13, the HVR-L3 comprises the aminoacid sequence of SEQ ID NO: 17, the HVR-H1 comprises the amino acidsequence of SEQ ID NO: 21, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO: 25, and the HVR-H3 comprises the amino acid sequence ofSEQ ID NO: 29; or (e) the HVR-L1 comprises the amino acid sequence ofSEQ ID NO: 8, the HVR-L2 comprises the amino acid sequence of SEQ ID NO:12, the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 21, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 24, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 28; or (f) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 172, the HVR-L2comprises the amino acid sequence of SEQ ID NO: 174, the HVR-L3comprises the amino acid sequence of SEQ ID NO: 176, the HVR-H1comprises the amino acid sequence of SEQ ID NO: 178, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 180, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 182; or (g) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 173, the HVR-L2comprises the amino acid sequence of SEQ ID NO: 175, the HVR-L3comprises the amino acid sequence of SEQ ID NO: 177, the HVR-H1comprises the amino acid sequence of SEQ ID NO: 179, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 181, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 183. In some embodimentsthat may be combined with any of the preceding embodiments, theanti-Siglec-9 antibody comprises a light chain variable domain and aheavy chain variable domain, wherein the light chain variable domaincomprises: an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 6,an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and whereinthe heavy chain variable region comprises: an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 19, an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 22, and an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 26. In some embodiments that may be combined withany of the preceding embodiments, the anti-Siglec-9 antibody comprises alight chain variable domain and a heavy chain variable domain, whereinthe light chain variable domain comprises: an HVR-L1 comprising theamino acid sequence of SEQ ID NO: 7, an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 11, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 15, and wherein the heavy chain variable regioncomprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:20, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, andan HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody comprises a light chain variable domain and aheavy chain variable domain, wherein the light chain variable domaincomprises: an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8,an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 12, and anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 16, and whereinthe heavy chain variable region comprises: an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 21, an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 24, and an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 28. In some embodiments that may be combined withany of the preceding embodiments, the anti-Siglec-9 antibody comprises alight chain variable domain and a heavy chain variable domain, whereinthe light chain variable domain comprises: an HVR-L1 comprising theamino acid sequence of SEQ ID NO: 9, an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 13, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 17, and wherein the heavy chain variable regioncomprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:21, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 25, andan HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody comprises a light chain variable domain and aheavy chain variable domain, wherein the light chain variable domaincomprises: an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8,an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 12, and anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 18, and whereinthe heavy chain variable region comprises: an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 21, an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 24, and an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 28. In some embodiments that may be combined withany of the preceding embodiments, the anti-Siglec-9 antibody comprises alight chain variable domain and a heavy chain variable domain, whereinthe light chain variable domain comprises: an HVR-L1 comprising theamino acid sequence of SEQ ID NO: 172, an HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 174, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 176, and wherein the heavy chain variable regioncomprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:178, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 180, andan HVR-H3 comprising the amino acid sequence of SEQ ID NO: 182. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody comprises a light chain variable domain and aheavy chain variable domain, wherein the light chain variable domaincomprises: an HVR-L1 comprising the amino acid sequence of SEQ ID NO:173, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 175, andan HVR-L3 comprising the amino acid sequence of SEQ ID NO: 177, andwherein the heavy chain variable region comprises: an HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 179, an HVR-H2 comprising theamino acid sequence of SEQ ID NO: 181, and an HVR-H3 comprising theamino acid sequence of SEQ ID NO: 183. In some embodiments that may becombined with any of the preceding embodiments, the anti-Siglec-9antibody comprises a light chain variable domain and a heavy chainvariable domain, wherein the light chain variable domain comprises: (a)an HVR-L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6-9, 172, and 173, or an amino acid sequencewith at least about 90% homology to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 6-9, 172, and 173; (b) an HVR-L2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 10-13, 174, and 175, or an amino acid sequence with at leastabout 90% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-13, 174, and 175; and (c) an HVR-L3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 14-18, 176, and 177, or an amino acid sequence with at leastabout 90% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14-18, 176, and 177; and wherein the heavychain variable domain comprises: (a) an HVR-H1 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 19-21, 178,and 179, or an amino acid sequence with at least about 90% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:19-21, 178, and 179; (b) an HVR-H2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22-25, 180, and 181,or an amino acid sequence with at least about 90% homology to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 22-25,180, and 181; and (c) an HVR-H3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 26-29, 182, and 183,or an amino acid sequence with at least about 90% homology to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 26-29,182, and 183. In some embodiments that may be combined with any of thepreceding embodiments, the anti-Siglec-9 antibody comprises a lightchain variable domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 61-115 and 197-204; and/or a heavychain variable domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 116-170 and 205-212. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody comprises a light chain variable domain and aheavy chain variable domain, wherein: (a) the light chain variabledomain comprises the amino acid sequence of SEQ ID NO: 61; and the heavychain variable domain comprises the amino acid sequence of SEQ ID NO:116; or (b) the light chain variable domain comprises the amino acidsequence of SEQ ID NO: 72; and the heavy chain variable domain comprisesthe amino acid sequence of SEQ ID NO: 127; or (c) the light chainvariable domain comprises the amino acid sequence of SEQ ID NO: 83; andthe heavy chain variable domain comprises the amino acid sequence of SEQID NO: 138; or (d) the light chain variable domain comprises the aminoacid sequence of SEQ ID NO: 94; and the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO: 149; or (e) the lightchain variable domain comprises the amino acid sequence of SEQ ID NO:105; and the heavy chain variable domain comprises the amino acidsequence of SEQ ID NO: 160; or (f) the light chain variable domaincomprises the amino acid sequence of SEQ ID NO: 197; and the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO: 205; or(g) the light chain variable domain comprises the amino acid sequence ofSEQ ID NO: 201; and the heavy chain variable domain comprises the aminoacid sequence of SEQ ID NO: 210. In some embodiments that may becombined with any of the preceding embodiments, the anti-Siglec-9antibody comprises a light chain variable domain of a monoclonalantibody selected from the group consisting of: 2D4, 2D5, 5B1, 6B2, 6D8,7H12, 5C6, 12B12, and 17C2; and/or a heavy chain variable domain of amonoclonal antibody selected from the group consisting of: 2D4, 2D5,5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2.

Other aspects of the present disclosure relate to an isolated (e.g.,monoclonal) anti-Siglec-9 antibody, wherein the anti-Siglec-9 antibodybinds to one or more amino acids within amino acid residues 20-347,20-140, 141-347, 146-347, 146-229, 236-336, or 146-347 of SEQ ID NO: 1;or within amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 20-347, 20-140, 141-347, 146-347,146-229, 236-336, or 146-347 of SEQ ID NO: 1. In some embodiments, theanti-Siglec-9 antibody binds to one or more amino acids within aminoacid residues selected from the group consisting of: i. amino acidresidues 62-76 of SEQ ID NO: 1, or amino acid residues on a mammalianSiglec-9 protein corresponding to amino acid residues 62-76 of SEQ IDNO: 1; ii. amino acid residues 62-76 and 86-92 of SEQ ID NO: 1, or aminoacid residues on a mammalian Siglec-9 protein corresponding to aminoacid residues 62-76 and 86-92 of SEQ ID NO: 1; iii. amino acid residues86-92 of SEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9protein corresponding to amino acid residues 86-92 of SEQ ID NO: 1; iv.amino acid residues 86-96 of SEQ ID NO: 1, or amino acid residues on amammalian Siglec-9 protein corresponding to amino acid residues 86-96 ofSEQ ID NO: 1; v. amino acid residues 86-96 and 105-116 of SEQ ID NO: 1,or amino acid residues on a mammalian Siglec-9 protein corresponding toamino acid residues 86-96 and 105-116 of SEQ ID NO: 1; vi. amino acidresidues 105-116 of SEQ ID NO: 1, or amino acid residues on a mammalianSiglec-9 protein corresponding to amino acid residues 105-116 of SEQ IDNO: 1; vii. amino acid residues 107-115 of SEQ ID NO: 1, or amino acidresidues on a mammalian Siglec-9 protein corresponding to amino acidresidues 107-115 of SEQ ID NO: 1; and viii. amino acid residues 185-194of SEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 185-194 of SEQ ID NO: 1. In someembodiments, the anti-Siglec-9 antibody binds to one or more amino acidswithin amino acid residues 62-76 of SEQ ID NO: 1, or amino acid residueson a mammalian Siglec-9 protein corresponding to amino acid residues62-76 of SEQ ID NO: 1. In some embodiments, the anti-Siglec-9 antibodybinds to one or more amino acids within amino acid residues 62-76 and86-92 of SEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9protein corresponding to amino acid residues 62-76 and 86-92 of SEQ IDNO: 1. In some embodiments, the anti-Siglec-9 antibody binds to one ormore amino acids within amino acid residues 86-92 of SEQ ID NO: 1, oramino acid residues on a mammalian Siglec-9 protein corresponding toamino acid residues 86-92 of SEQ ID NO: 1. In some embodiments, theanti-Siglec-9 antibody binds to one or more amino acids within aminoacid residues 86-96 of SEQ ID NO: 1, or amino acid residues on amammalian Siglec-9 protein corresponding to amino acid residues 86-96 ofSEQ ID NO: 1. In some embodiments, the anti-Siglec-9 antibody binds toone or more amino acids within amino acid residues 86-96 and 105-116 ofSEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 86-96 and 105-116 of SEQ ID NO: 1.In some embodiments, the anti-Siglec-9 antibody binds to one or moreamino acids within amino acid residues 105-116 of SEQ ID NO: 1, or aminoacid residues on a mammalian Siglec-9 protein corresponding to aminoacid residues 105-116 of SEQ ID NO: 1. In some embodiments, theanti-Siglec-9 antibody binds to one or more amino acids within aminoacid residues 107-115 of SEQ ID NO: 1, or amino acid residues on amammalian Siglec-9 protein corresponding to amino acid residues 107-115of SEQ ID NO: 1. In some embodiments, the anti-Siglec-9 antibody bindsto one or more amino acids within amino acid residues 185-194 of SEQ IDNO: 1, or amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 185-194 of SEQ ID NO: 1. Otheraspects of the present disclosure relate to an isolated anti-Siglec-9antibody, wherein the anti-Siglec-9 antibody binds to one or more aminoacid residues selected from the group consisting of L22, H48, W50, I51,Y52, K123, I126, D189, P190, R194 of SEQ ID NO: 1, or one or more aminoacid residues on a mammalian Siglec-7 protein corresponding to an aminoacid residue selected from the group consisting of L22, H48, W50, I51,Y52, K123, I126, D189, P190, R194 of SEQ ID NO: 1.

Other aspects of the present disclosure relate to an isolatedanti-Siglec-9 antibody, wherein the anti-Siglec-9 antibody binds to oneor more amino acids within amino acid residues 62-76 of SEQ ID NO: 1, oramino acid residues on a mammalian Siglec-9 protein corresponding toamino acid residues 62-76 of SEQ ID NO: 1. Other aspects of the presentdisclosure relate to an isolated anti-Siglec-9 antibody, wherein theanti-Siglec-9 antibody binds to one or more amino acids within aminoacid residues 62-76 and 86-92 of SEQ ID NO: 1, or amino acid residues ona mammalian Siglec-9 protein corresponding to amino acid residues 62-76and 86-92 of SEQ ID NO: 1. Other aspects of the present disclosurerelate to an isolated anti-Siglec-9 antibody, wherein the anti-Siglec-9antibody binds to one or more amino acids within amino acid residues86-92 of SEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9protein corresponding to amino acid residues 86-92 of SEQ ID NO: 1.Other aspects of the present disclosure relate to an isolatedanti-Siglec-9 antibody, wherein the anti-Siglec-9 antibody binds to oneor more amino acids within amino acid residues 86-96 of SEQ ID NO: 1, oramino acid residues on a mammalian Siglec-9 protein corresponding toamino acid residues 86-96 of SEQ ID NO: 1. Other aspects of the presentdisclosure relate to an isolated anti-Siglec-9 antibody, wherein theanti-Siglec-9 antibody binds to one or more amino acids within aminoacid residues 86-96 and 105-116 of SEQ ID NO: 1, or amino acid residueson a mammalian Siglec-9 protein corresponding to amino acid residues86-96 and 105-116 of SEQ ID NO: 1. Other aspects of the presentdisclosure relate to an isolated anti-Siglec-9 antibody, wherein theanti-Siglec-9 antibody binds to one or more amino acids within aminoacid residues 105-116 of SEQ ID NO: 1, or amino acid residues on amammalian Siglec-9 protein corresponding to amino acid residues 105-116of SEQ ID NO: 1. Other aspects of the present disclosure relate to anisolated anti-Siglec-9 antibody, wherein the anti-Siglec-9 antibodybinds to one or more amino acids within amino acid residues 107-115 ofSEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 107-115 of SEQ ID NO: 1. Otheraspects of the present disclosure relate to an isolated anti-Siglec-9antibody, wherein the anti-Siglec-9 antibody binds to one or more aminoacids within amino acid residues 185-194 of SEQ ID NO: 1, or amino acidresidues on a mammalian Siglec-9 protein corresponding to amino acidresidues 185-194 of SEQ ID NO: 1.

Other aspects of the present disclosure relate to an isolated (e.g.,monoclonal) anti-Siglec-9 antibody, wherein the anti-Siglec-9 antibodycomprises a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain, the heavy chainvariable domain, or both comprise at least one, two, three, four, five,or six HVRs selected from HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, andHVR-H3 of a monoclonal antibody selected from the group consisting of:2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2. In someembodiments: (a) the HVR-L1 comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 6-9, 172, and 173; or (b) theHVR-L2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-13, 174, and 175; or (c) the HVR-L3comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 14-18, 176, and 177; or (d) the HVR-H1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 19-21,178, and 179; or (e) the HVR-H2 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22-25, 180, and 181;or (f) the HVR-H3 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 26-29, 182, and 183. In someembodiments: (a) the HVR-L1 comprises the amino acid sequence of SEQ IDNO: 6, the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 10,the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 14, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 19, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 22, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 26; or (b) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 7, the HVR-L2 comprisesthe amino acid sequence of SEQ ID NO: 11, the HVR-L3 comprises the aminoacid sequence of SEQ ID NO: 15, the HVR-H1 comprises the amino acidsequence of SEQ ID NO: 20, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO: 23, and the HVR-H3 comprises the amino acid sequence ofSEQ ID NO: 27; or (c) the HVR-L1 comprises the amino acid sequence ofSEQ ID NO: 8, the HVR-L2 comprises the amino acid sequence of SEQ ID NO:12, the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 16, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 21, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 24, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 28; or (d) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 9, the HVR-L2 comprisesthe amino acid sequence of SEQ ID NO: 13, the HVR-L3 comprises the aminoacid sequence of SEQ ID NO: 17, the HVR-H1 comprises the amino acidsequence of SEQ ID NO: 21, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO: 25, and the HVR-H3 comprises the amino acid sequence ofSEQ ID NO: 29; or (c) the HVR-L1 comprises the amino acid sequence ofSEQ ID NO: 8, the HVR-L2 comprises the amino acid sequence of SEQ ID NO:12, the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 21, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 24, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 28; or (f) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 172, the HVR-L2comprises the amino acid sequence of SEQ ID NO: 174, the HVR-L3comprises the amino acid sequence of SEQ ID NO: 176, the HVR-H1comprises the amino acid sequence of SEQ ID NO: 178, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 180, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 182; or (g) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 173, the HVR-L2comprises the amino acid sequence of SEQ ID NO: 175, the HVR-L3comprises the amino acid sequence of SEQ ID NO: 177, the HVR-H1comprises the amino acid sequence of SEQ ID NO: 179, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 181, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 183. In someembodiments, the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 6, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 10, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14, and wherein the heavy chain variable domain comprises: an HVR-H1comprising the amino acid sequence of SEQ ID NO: 19, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 22, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 7, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 11, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:15, and wherein the heavy chain variable domain comprises: an HVR-H1comprising the amino acid sequence of SEQ ID NO: 20, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 23, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 8, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 12, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:16, and wherein the heavy chain variable domain comprises: an HVR-H1comprising the amino acid sequence of SEQ ID NO: 21, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 24, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 28. In someembodiments, the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 9, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 13, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:17, and wherein the heavy chain variable domain comprises: an HVR-H1comprising the amino acid sequence of SEQ ID NO: 21, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 25, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 29. In someembodiments, the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 8, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 12, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:18, and wherein the heavy chain variable domain comprises: an HVR-H1comprising the amino acid sequence of SEQ ID NO: 21, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 24, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 28. In someembodiments, the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 172, an HVR-L2 comprising the amino acid sequence of SEQID NO: 174, and an HVR-L3 comprising the amino acid sequence of SEQ IDNO: 176, and wherein the heavy chain variable domain comprises: anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 178, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 180, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 182. In someembodiments, the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 173, an HVR-L2 comprising the amino acid sequence of SEQID NO: 175, and an HVR-L3 comprising the amino acid sequence of SEQ IDNO: 177, and wherein the heavy chain variable domain comprises: anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 179, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 181, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 183. In someembodiments, the light chain variable domain comprises: (a) an HVR-L1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 6-9, 172, and 173, or an amino acid sequence with at leastabout 90% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6-9, 172, and 173; (b) an HVR-L2 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:10-13, 174, and 175, or an amino acid sequence with at least about 90%homology to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 10-13, 174, and 175; and (c) an HVR-L3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 14-18,176, and 177, or an amino acid sequence with at least about 90% homologyto an amino acid sequence selected from the group consisting of SEQ IDNOs: 14-18, 176, and 177; and wherein the heavy chain variable domaincomprises: (a) an HVR-H1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 19-21, 178, and 179, or an aminoacid sequence with at least about 90% homology to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19-21, 178, and 179;(b) an HVR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22-25, 180, and 181, or an amino acid sequencewith at least about 90% homology to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 22-25, 180, and 181; and (c) anHVR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 26-29, 182, and 183, or an amino acid sequencewith at least about 90% homology to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 26-29, 182, and 183.

Other aspects of the present disclosure relate to an isolatedanti-Siglec-9 antibody, wherein the anti-Siglec-9 antibody comprises alight chain variable domain and a heavy chain variable domain, whereinthe light chain variable domain comprises: an HVR-L1 comprising theamino acid sequence of SEQ ID NO: 6, an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 10, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 14, and wherein the heavy chain variable domaincomprises: an HVR-H1 comprising the amino acid sequence of SEQ ID NO:19, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, andan HVR-H3 comprising the amino acid sequence of SEQ ID NO: 26. Otheraspects of the present disclosure relate to an isolated anti-Siglec-9antibody, wherein the anti-Siglec-9 antibody comprises a light chainvariable domain and a heavy chain variable domain, wherein the lightchain variable domain comprises: an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 7, an HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 11, and an HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 15, and wherein the heavy chain variable domain comprises: anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 23, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 27. Other aspects ofthe present disclosure relate to an isolated anti-Siglec-9 antibody,wherein the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 8, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 12, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:16, and wherein the heavy chain variable domain comprises: an HVR-H1comprising the amino acid sequence of SEQ ID NO: 21, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 24, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 28. Other aspects ofthe present disclosure relate to an isolated anti-Siglec-9 antibody,wherein the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 9, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 13, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:17, and wherein the heavy chain variable domain comprises: an HVR-H1comprising the amino acid sequence of SEQ ID NO: 21, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 25, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 29. Other aspects ofthe present disclosure relate to an isolated anti-Siglec-9 antibody,wherein the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 8, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 12, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:18, and wherein the heavy chain variable domain comprises: an HVR-H1comprising the amino acid sequence of SEQ ID NO: 21, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 24, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 28. Other aspects ofthe present disclosure relate to an isolated anti-Siglec-9 antibody,wherein the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 172, an HVR-L2 comprising the amino acid sequence of SEQID NO: 174, and an HVR-L3 comprising the amino acid sequence of SEQ IDNO: 176, and wherein the heavy chain variable domain comprises: anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 178, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 180, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 182. Other aspects ofthe present disclosure relate to an isolated anti-Siglec-9 antibody,wherein the anti-Siglec-9 antibody comprises a light chain variabledomain and a heavy chain variable domain, wherein the light chainvariable domain comprises: an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 173, an HVR-L2 comprising the amino acid sequence of SEQID NO: 175, and an HVR-L3 comprising the amino acid sequence of SEQ IDNO: 177, and wherein the heavy chain variable domain comprises: anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 179, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 181, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 183. Other aspects ofthe present disclosure relate to an isolated (e.g., monoclonal)anti-Siglec-9 antibody, wherein the anti-Siglec-9 antibody comprises alight chain variable domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 61-115 and 197-204 and/or aheavy chain variable domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 116-170 and 205-212. Otheraspects of the present disclosure relate to an isolated (e.g.,monoclonal) anti-Siglec-9 antibody, wherein the anti-Siglec-9 antibodycomprises a light chain variable domain and a heavy chain variabledomain, and wherein: (a) the light chain variable domain comprises theamino acid sequence of SEQ ID NO: 61; and the heavy chain variabledomain comprises the amino acid sequence of SEQ ID NO: 116; or (b) thelight chain variable domain comprises the amino acid sequence of SEQ IDNO: 72; and the heavy chain variable domain comprises the amino acidsequence of SEQ ID NO: 127; or (c) the light chain variable domaincomprises the amino acid sequence of SEQ ID NO: 83; and the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO: 138; or(d) the light chain variable domain comprises the amino acid sequence ofSEQ ID NO: 94; and the heavy chain variable domain comprises the aminoacid sequence of SEQ ID NO: 149; or (e) the light chain variable domaincomprises the amino acid sequence of SEQ ID NO: 105; and the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO: 160; or(f) the light chain variable domain comprises the amino acid sequence ofSEQ ID NO: 197; and the heavy chain variable domain comprises the aminoacid sequence of SEQ ID NO: 205; or (g) the light chain variable domaincomprises the amino acid sequence of SEQ ID NO: 201; and the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO: 210.Other aspects of the present disclosure relate to an isolated (e.g.,monoclonal) anti-Siglec-9 antibody, wherein the anti-Siglec-9 antibodycomprises a light chain variable domain of a monoclonal antibodyselected from the group consisting of: 2D4, 2D5, 5B1, 6B2, 6D8, 7H12,5C6, 12B12, and 17C2; and/or a heavy chain variable domain of amonoclonal antibody selected from the group consisting of: 2D4, 2D5,5B1, 6B2, 6D8, 7H12, 5C6, 12B 12, and 17C2. Other aspects of the presentdisclosure relate to an isolated (e.g., monoclonal) anti-Siglec-9antibody, wherein the anti-Siglec-9 antibody competes with one or moreantibodies selected from the group consisting of 2D4, 2D5, 5B1, 6B2,6D8, 7H12, 5C6, 12B12, 17C2, and any combination thereof for binding toSiglec-9. Other aspects of the present disclosure relate to an isolated(e.g., monoclonal) anti-Siglec-9 antibody which binds essentially thesame Siglec-9 epitope as a monoclonal antibody selected from the groupconsisting of: 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2.Other aspects of the present disclosure relate to an isolated (e.g.,monoclonal) anti-Siglec-9 antibody, wherein the anti-Siglec-9 antibodycomprises a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain comprises: (a) an HVR-L1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 6-9, 172, and 173, or an amino acid sequence with at leastabout 90% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6-9, 172, and 173; (b) an HVR-L2 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:10-13, 174, and 175, or an amino acid sequence with at least about 90%homology to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 10-13, 174, and 175; and (c) an HVR-L3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 14-18,176, and 177, or an amino acid sequence with at least about 90% homologyto an amino acid sequence selected from the group consisting of SEQ IDNOs: 14-18, 176, and 177; and wherein the heavy chain variable domaincomprises: (a) an HVR-H1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 19-21, 178, and 179, or an aminoacid sequence with at least about 90% homology to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19-21, 178, and 179;(b) an HVR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22-25, 180, and 181, or an amino acid sequencewith at least about 90% homology to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 22-25, 180, and 181; and (c) anHVR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 26-29, 182, and 183, or an amino acid sequencewith at least about 90% homology to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 26-29, 182, and 183.

In some embodiments that may be combined with any of the precedingembodiments, the antibody is of the IgG class the IgM class, or the IgAclass. In some embodiments that may be combined with any of thepreceding embodiments, the anti-Siglec-9 antibody has an IgG 1, IgG2,IgG3, or IgG4 isotype. In some embodiments that may be combined with anyof the preceding embodiments, the antibody binds an inhibitory Fcreceptor. In some embodiments that may be combined with any of thepreceding embodiments, the inhibitory Fc receptor is inhibitory Fc-gammareceptor IIB (FcγIIB). In some embodiments that may be combined with anyof the preceding embodiments: (a) the anti-Siglec-9 antibody has a humanor mouse IgG1 isotype and comprises one or more amino acid substitutionsin the Fc region at a residue position selected from the groupconsisting of: N297A, D265A, D270A, L234A, L235A, G237A, P238D, L328E,E233D, G237D, H268D, P271G, A330R, C226S, C229S, E233P, L234V, L234F,L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S,P238A, A327Q, A327G, P329A, K322A, T394D, and any combination thereof,wherein the numbering of the residues is according to EU or Kabatnumbering, or comprises an amino acid deletion in the Fc region at aposition corresponding to glycine 236; (b) the anti-Siglec-9 antibodyhas an IgG1 isotype and comprises an IgG2 isotype heavy chain constantdomain 1(CH1) and hinge region, optionally wherein the IgG2 isotype CH1and hinge region comprises the amino acid sequence of ASTKGPSVFPLAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 171), andoptionally wherein the antibody Fc region comprises a S267E amino acidsubstitution, a L328F amino acid substitution, or both, and/or a N297Aor N297Q amino acid substitution, wherein the numbering of the residuesis according to EU numbering; (c) the anti-Siglec-9 antibody has an IgG2isotype and comprises one or more amino acid substitutions in the Fcregion at a residue position selected from the group consisting of:P238S, V234A, G237A, H268A, H268Q, V309L, A330S, P331 S, C214S, C232S,C233S, S267E, L328F, M252Y, S254T, T256E, H268E, N297A, N297Q, A330L,and any combination thereof, wherein the numbering of the residues isaccording to EU or Kabat numbering; (d) the anti-Siglec-9 antibody has ahuman or mouse IgG4 isotype and comprises one or more amino acidsubstitutions in the Fc region at a residue position selected from thegroup consisting of: L235A, G237A, S228P, L236E, S267E, E318A, L328F,M252Y, S254T, T256E, E233P, F234V, L234A/F234A, S228P, S241P, L248E,T394D, N297A, N297Q, L235E, and any combination thereof, wherein thenumbering of the residues is according to EU or Kabat numbering; or (e)the anti-Siglec-9 antibody has a hybrid IgG2/4 isotype, and optionallywherein the antibody comprises an amino acid sequence comprising aminoacids 118 to 260 of human IgG2 and amino acids 261 to 447 of human IgG4,wherein the numbering of the residues is according to EU or, Kabatnumbering. In some embodiments that may be combined with any of thepreceding embodiments: (a) the anti-Siglec-9 antibody has a human ormouse IgG1 isotype and comprises one or more amino acid substitutions inthe Fc region at a residue position selected from the group consistingof: N297A, N297Q, D270A, D265A, L234A, L235A, C226S, C229S, P238S,E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S,T394D, A330L, M252Y, S254T, T256E, and any combination thereof, whereinthe numbering of the residues is according to EU or Kabat numbering; (b)the anti-Siglec-9 antibody has an IgG2 isotype and comprises one or moreamino acid substitutions in the Fc region at a residue position selectedfrom the group consisting of: P238S, V234A, G237A, H268A, H268Q, H268E,V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E,and any combination thereof, wherein the numbering of the residues isaccording to EU or Kabat numbering; or (c) the anti-Siglec-9 antibodyhas an IgG4 isotype and comprises one or more amino acid substitutionsin the Fc region at a residue position selected from the groupconsisting of: E233P, F234V, L234A/F234A, L235A, G237A, E318A, S228P,L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and anycombination thereof, wherein the numbering of the residues is accordingto EU or Kabat numbering. In some embodiments that may be combined withany of the preceding embodiments: (a) the Fc region further comprisesone or more additional amino acid substitutions at a position selectedfrom the group consisting of A330L, L234F; L235E, P331S, and anycombination thereof, wherein the numbering of the residues is accordingto EU or Kabat numbering; (b) the Fc region further comprises one ormore additional amino acid substitutions at a position selected from thegroup consisting of M252Y, S254T, T256E, and any combination thereof,wherein the numbering of the residues is according to EU or Kabatnumbering; or (c) the Fc region further comprises a S228P amino acidsubstitution according to EU or Kabat numbering. In some embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas an IgG4 isotype. n some embodiments that may be combined with any ofthe preceding embodiments, the anti-Siglec-9 antibody comprises an S228Pamino acid substitution at residue position 228, an F234A amino acidsubstitution at residue position 234, and an L235A amino acidsubstitution at residue position 235, wherein the numbering of theresidue position is according to EU or Kabat numbering. In someembodiments that may be combined with any of the preceding embodiments,the Siglec-9 protein is a mammalian protein or a human protein. In someembodiments that may be combined with any of the preceding embodiments,the Siglec-9 protein is a wild-type protein. In some embodiments thatmay be combined with any of the preceding embodiments, the Siglec-9protein is a naturally occurring variant. In some embodiments that maybe combined with any of the preceding embodiments, the Siglec-9 proteinis expressed on one or more cells selected from the group consisting ofhuman dendritic cells, human macrophages, human neutrophils, human NKcells, human monocytes, human osteoclasts, human T cells, human T helpercell, human cytotoxic T cells, human granulocytes, and human microglia.In some embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody hinds specifically to amammalian Siglec-9 protein, human Siglec-9 protein, or both. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody binds Siglec-9 in a pH dependent manner. Insome embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody binds Siglec-9 at a pH thatranges from 5.5 to 8.0. In some embodiments that may be combined withany of the preceding embodiments, the anti-Siglec-9 antibody dissociatesfrom Siglec-9 at a pH of less than 5.0. In some embodiments that may becombined with any of the preceding embodiments, the anti-Siglec-9antibody is an antibody fragment that binds to an epitope comprisingamino acid residues on human Siglec-9 or a mammalian Siglec-9 protein.In some embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody is an antibody fragment thatbinds to one or more human proteins selected from the group consistingof human Siglec-9, a naturally occurring variant of human Siglec-9, anda disease variant of human Siglec-9. In some embodiments that may becombined with any of the preceding embodiments, the antibody fragment iscross-linked to a second antibody fragment that binds to one or morehuman proteins selected from the group consisting of human Siglec-9, anaturally occurring variant of human Siglec-9, and a disease variant ofhuman Siglec-9. In some embodiments that may be combined with any of thepreceding embodiments, the fragment is an Fab, Fab′, Fab′-SH, F(ab′)2,Fv, or scFv fragment. In some embodiments that may be combined with anyof the preceding embodiments, the anti-Siglec-9 antibody is a murineantibody. In some embodiments that may be combined with any of thepreceding embodiments, the anti-Siglec-9 antibody is a humanizedantibody, a bispecific antibody, a monoclonal antibody, a multivalentantibody, a conjugated antibody, or a chimeric antibody. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody is a bispecific antibody recognizing a firstantigen and a second antigen. In some embodiments that may be combinedwith any of the preceding embodiments, the first antigen is Siglec-9 andthe second antigen is: (a) an antigen facilitating transport across theblood-brain-barrier; (b) an antigen facilitating transport across theblood-brain-barrier selected from the group consisting of transferrinreceptor (TR), insulin receptor (HIR), insulin-like growth factorreceptor (IGFR), low-density lipoprotein receptor related proteins 1 and2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, a llama singledomain antibody, TMEM 30(A), a protein transduction domain, TAT, Syn-B,penetratin, a poly-arginine peptide, an angiopep peptide, and ANG1005;(c) a disease-causing agent selected from the group consisting ofdisease-causing peptides or proteins or, disease-causing nucleic acids,wherein the disease-causing nucleic acids are antisense GGCCCC (G2C4)repeat-expansion RNA, the disease-causing proteins are selected from thegroup consisting of amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides; (d) ligands and/or proteins expressed on immunecells, wherein the ligands and/or proteins selected from the groupconsisting of PD1/PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR,PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT,VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39,CD73, and phosphatidylserine; and (e) a protein, lipid, polysaccharide,or glycolipid expressed on one or more tumor cells. In some embodimentsthat may be combined with any of the preceding embodiments, theanti-Siglec-9 antibody is a conjugated antibody. In some embodimentsthat may be combined with any of the preceding embodiments, theanti-Siglec-9 antibody is conjugated to a detectable marker, a toxin, ora therapeutic agent. In some embodiments that may be combined with anyof the preceding embodiments, the anti-Siglec-9 antibody is conjugatedto a toxin selected from the group consisting of ricin, ricin A-chain,doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin,curicin, crotin, calicheanmicin, Saponaria officinalis inhibitor,glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin,duocarmycins, dolastatin, cc1065, and a cisplatin. In some embodimentsthat may be combined with any of the preceding embodiments, theanti-Siglec-9 antibody is used in combination with one or moreantibodies that specifically bind a disease-causing protein selectedfrom the group consisting of amyloid beta, oligomeric amyloid beta,amyloid beta plaques, amyloid precursor protein or fragments thereof,Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9open reading frame 72), prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides, and any combination thereof; or with one or moreantibodies that bind an immunomodulatory protein selected from the groupconsisting of: PD1/PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27,GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30,TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5,CD39, CD73, TREM1, TREM2, CD33, Siglec-5, Siglec-7, Siglec-11,phosphatidylserine, disease-causing nucleic acids, antisense GGCCCC(G2C4) repeat-expansion RNA, and any combination thereof. In someembodiments that may be combined with any of the preceding embodiments,the anti-Siglec-9 antibody has dissociation constant (K_(D)) for humanSiglec-9 and mammalian Siglec-9 that ranges from about 10 nM to about 10pM, or less than 10 pM, wherein the K_(D) is determined at a temperatureof approximately 25° C. In some embodiments that may be combined withany of the preceding embodiments, the anti-Siglec-9 antibody hasdissociation constant (K_(D)) for human Siglec-9 that ranges from about9 nM to about 300 pM, or less than 300 pM, wherein the K_(D) isdetermined at a temperature of approximately 25° C. In some embodimentsthat may be combined with any of the preceding embodiments, theanti-Siglec-9 antibody has dissociation constant (K_(D)) for humanSiglec-9 that ranges from about 9 nM to about 230 pM, or less than 230pM, wherein the K_(D) is determined at a temperature of approximately25° C.

Other aspects of the present disclosure relate to an isolated nucleicacid comprising a nucleic acid sequence encoding the anti-Siglec-9antibody of any of the preceding embodiments. Other aspects of thepresent disclosure relate to a vector comprising the nucleic acid of anyof the preceding embodiments. Other aspects of the present disclosurerelate to an isolated host cell comprising the vector of any of thepreceding embodiments. Other aspects of the present disclosure relate toa method of producing an anti-Siglec-9 antibody, comprising culturingthe host cell of any of the preceding embodiments so that theanti-Siglec-9 antibody is produced. In some embodiments, the methodfurther comprises recovering the anti-Siglec-9 antibody produced by thehost cell. Other aspects of the present disclosure relate to an isolatedanti-Siglec-9 antibody produced by the method of any of the precedingembodiments. Other aspects of the present disclosure relate to apharmaceutical composition comprising the anti-Siglec-9 antibody of anyof the preceding embodiments, and a pharmaceutically acceptable carrier.

Other aspects of the present disclosure relate to a method ofpreventing, reducing risk, or treating a disease, disorder, or injuryselected from the group consisting of dementia, frontotemporal dementia,Alzheimer's disease, vascular dementia, mixed dementia,Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophiclateral sclerosis, Huntington's disease, taupathy disease, Nasu-Hakoladisease, stroke, acute trauma, chronic trauma, lupus, acute and chroniccolitis, rheumatoid arthritis, wound healing, Crohn's disease,inflammatory bowel disease, ulcerative colitis, obesity, malaria,essential tremor, central nervous system lupus, Behcet's disease,Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,Shy-Drager syndrome, progressive supranuclear palsy, cortical basalganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, cyc infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, andcancer, bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,tumors that express one or more Siglec-9 ligands, thyroid cancer,infections, CNS herpes, parasitic infections, Trypanosome infection,Cruzi infection, Pseudomonas aeruginosa infection, Leishmania donovaniinfection, group B Streptococcus infection, Campylobacter jejuniinfection, Neisseria meningiditis infection, type I HIV, and Haemophilusinfluenza, comprising administering to an individual in need thereof atherapeutically effective amount of an agent that decreases cellularlevels of Siglec-9, inhibits interaction between Siglec-9 and one ormore Siglec-9 ligands, or both. Other aspects of the present disclosurerelate to an agent that decreases cellular levels of Siglec-9, inhibitsinteraction between Siglec-9 and one or more Siglec-9 ligands, or bothfor use in preventing, reducing risk, or treating a disease, disorder,or injury selected from the group consisting of dementia, frontotemporaldementia, Alzheimer's disease, vascular dementia, mixed dementia,Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophiclateral sclerosis, Huntington's disease, taupathy disease, Nasu-Hakoladisease, stroke, acute trauma, chronic trauma, lupus, acute and chroniccolitis, rheumatoid arthritis, wound healing, Crohn's disease,inflammatory bowel disease, ulcerative colitis, obesity, malaria,essential tremor, central nervous system lupus, Behcet's disease,Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,Shy-Drager syndrome, progressive supranuclear palsy, cortical basalganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, andcancer, bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,tumors that express one or more Siglec-9 ligands, thyroid cancer,infections, CNS herpes, parasitic infections, Trypanosome infection,Cruzi infection, Pseudomonas aeruginosa infection, Leishmania donovaniinfection, group B Streptococcus infection, Campylobacter jejuniinfection, Neisseria meningiditis infection, type I HIV, and Haemophilusinfluenza. Other aspects of the present disclosure relate to use of anagent that decreases cellular levels of Siglec-9, inhibits interactionbetween Siglec-9 and one or more Siglec-9 ligands, or both in themanufacture of a medicament for preventing, reducing risk, or treating adisease, disorder, or injury selected from the group consisting ofdementia, frontotemporal dementia, Alzheimer's disease, vasculardementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressurehydrocephalus, amyotrophic lateral sclerosis, Huntington's disease,taupathy disease, Nasu-Hakola disease, stroke, acute trauma, chronictrauma, lupus, acute and chronic colitis, rheumatoid arthritis, woundhealing, Crohn's disease, inflammatory bowel disease, ulcerativecolitis, obesity, malaria, essential tremor, central nervous systemlupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies,multiple system atrophy, Shy-Drager syndrome, progressive supranuclearpalsy, cortical basal ganglionic degeneration, acute disseminatedencephalomyelitis, granulomartous disorders, sarcoidosis, diseases ofaging, seizures, spinal cord injury, traumatic brain injury, age relatedmacular degeneration, glaucoma, retinitis pigmentosa, retinaldegeneration, respiratory tract infection, sepsis, eye infection,systemic infection, lupus, arthritis, multiple sclerosis, low bonedensity, osteoporosis, osteogenesis, osteopetrotic disease, Paget'sdisease of bone, and cancer, bladder cancer, brain cancer, breastcancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer,renal cell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma,non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovariancancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloidleukemia (CML), multiple myeloma, polycythemia vera, essentialthrombocytosis, primary or idiopathic myelofibrosis, primary oridiopathic myelosclerosis, myeloid-derived tumors, tumors that expressSiglec-9, tumors that express one or more Siglec-9 ligands, thyroidcancer, infections, CNS herpes, parasitic infections, Trypanosomeinfection, Cruzi infection, Pseudomonas aeruginosa infection, Leishmaniadonovani infection, group B Streptococcus infection, Campylobacterjejuni infection, Neisseria meningiditis infection, type I HIV, andHaemophilus influenza. Other aspects of the present disclosure relate toa method of preventing, reducing risk, or treating a disease, disorder,or injury selected from the group consisting of dementia, frontotemporaldementia, Alzheimer's disease, vascular dementia, mixed dementia,taupathy disease, infections, and cancer, comprising administering to anindividual in need thereof a therapeutically effective amount of anagent that decreases cellular levels of Siglec-9, inhibits interactionbetween Siglec-9 and one or more Siglec-9 ligands, or both. Otheraspects of the present disclosure relate to an agent that decreasescellular levels of Siglec-9, inhibits interaction between Siglec-9 andone or more Siglec-9 ligands, or both for use in preventing, reducingrisk, or treating a disease, disorder, or injury selected from the groupconsisting of dementia, frontotemporal dementia, Alzheimer's disease,vascular dementia, mixed dementia, taupathy disease, infections, andcancer. Other aspects of the present disclosure relate to use of anagent that decreases cellular levels of Siglec-9, inhibits interactionbetween Siglec-9 and one or more Siglec-9 ligands, or both in themanufacture of a medicament for preventing, reducing risk, or treating adisease, disorder, or injury selected from the group consisting ofdementia, frontotemporal dementia, Alzheimer's disease, vasculardementia, mixed dementia, taupathy disease, infections, and cancer. Insome embodiments the disease, disorder, or injury is cancer, and whereinthe agent inhibits one or more Siglec-9 activities selected from thegroup consisting of: (a) promoting proliferation, maturation, migration,differentiation, and/or functionality of one or more of immunosuppressordendritic cells, immunosuppressor macrophages, immunosuppressorneutrophils, immunosuppressor NK cells, myeloid derived suppressorcells, tumor-associated macrophages, tumor-associated suppressorneutrophils, tumor-associated suppressor NK cells, and regulatory Tcells; (b) enhancing infiltration of one or more of immunosuppressordendritic cells, immunosuppressor macrophages, immunosuppressorneutrophils, immunosuppressor NK cells, myeloid derived suppressorcells, tumor-associated macrophages, tumor-associated suppressorneutrophils, tumor-associated suppressor NK cells, and regulatory Tcells into tumors; (c) increasing number of tumor-promotingmyeloid/granulocytic immune-suppressive cells in a tumor, in peripheralblood, or other lymphoid organ; (d) enhancing tumor-promoting activityof myeloid-derived suppressor cells (MDSC); (e) increasing expression oftumor-promoting cytokines in a tumor or in peripheral blood, optionallywherein the tumor-promoting cytokines are TGF-beta or IL-10; (f)increasing tumor infiltration of tumor-promoting FoxP3+ regulatory Tlymphocytes; (g) decreasing activation of tumor-specific T lymphocyteswith tumor killing potential; (h) decreasing infiltration oftumor-specific T lymphocytes with tumor killing potential; (i)increasing infiltration of one or more of immunosuppressor dendriticcells, immunosuppressor macrophages, immunosuppressor neutrophils,immunosuppressor NK cells, myeloid-derived suppressor cells,tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, non-tumorigenic CD45⁺CD14⁺ myeloid cells, andregulatory T cells into tumors; (j) increasing the number oftumor-promoting myeloid/granulocytic immune-suppressive cells and/ornon-tumorigenic CD45⁺CD14⁺ myeloid cells in a tumor, in peripheralblood, or other lymphoid organ; (k) enhancing tumor-promoting activityof myeloid-derived suppressor cells and/or non-tumorigenic CD45⁺CD14⁺myeloid cells; (l) enhancing survival of non-tumorigenic myeloid-derivedsuppressor cells and/or non-tumorigenic CD45⁺CD14⁺ myeloid cells; (m)decreasing activation of tumor-specific T lymphocytes with tumor killingpotential; (n) decreasing infiltration of tumor-specific NK cells withtumor killing potential; (o) increasing tumor volume; (p) increasingtumor growth rate; (q) increasing metastasis; (r) increasing rate oftumor recurrence; (s) decreasing efficacy of one or moreimmune-therapies that modulate anti-tumor T cell responses, optionallywherein the one or more immune-therapies are immune-therapies thattarget one or more target proteins selected from the group consisting ofPD1/PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4,PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR,GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, and anycombination thereof, or one or more cancer vaccines; (t) inhibition ofPLCγ/PKC/calcium mobilization; and (u) inhibition of PI3K/Akt, Ras/MAPKsignaling. In some embodiments, the cancer expresses Siglec-9 or one ormore Siglec-9 ligands. In some embodiments that may be combined with anyof the preceding embodiments, the disease, disorder, or injury iscancer, and the agent inhibits one or more Siglec-9 activities selectedfrom the group consisting of: (a) promoting proliferation, maturation,migration, differentiation, and/or functionality of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, non-tumorigenic myeloid derived suppressorcells, tumor-associated macrophages, non-tumorigenic CD14⁺ myeloidcells, and regulatory T cells; (b) enhancing infiltration of one or moreof immunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, non-tumorigenic myeloid derived suppressorcells, tumor-associated macrophages, and regulatory T cells into tumors;(c) increasing number of tumor-promoting myeloid/granulocyticimmune-suppressive cells and/or non-tumorigenic CD14⁺ myeloid cells in atumor, in peripheral blood, or other lymphoid organ; (d) enhancingtumor-promoting activity of non-tumorigenic myeloid-derived suppressorcells and/or non-tumorigenic CD14⁺ myeloid cells; (e) increasingexpression of tumor-promoting cytokines in a tumor or in peripheralblood, optionally wherein the tumor-promoting cytokines are TGF-beta orIL-10; (f) increasing tumor infiltration of tumor-promoting FoxP3+regulatory T lymphocytes; (g) decreasing activation of tumor-specific Tlymphocytes with tumor killing potential; (h) decreasing infiltration oftumor-specific T lymphocytes with tumor killing potential; (i)decreasing infiltration of tumor-specific NK cells with tumor killingpotential; (j) decreasing tumor killing potential of NK cells; (k)decreasing infiltration of tumor-specific B lymphocytes with potentialto enhance immune response; (l) increasing tumor volume; (m) increasingtumor growth rate; (n) increasing metastasis; (o) increasing rate oftumor recurrence; (p) increasing expression of one or more PD-1 ligands;(q) decreasing efficacy of one or more immune-therapies that modulateanti-tumor T cell responses, optionally wherein the one or moreimmune-therapies are immune-therapies that target one or more proteinsselected from the group consisting of CD40, OX40, ICOS, CD28,CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM,BTLA, KIR, GAL9, TIM3, A2AR, LAG, DR-5, TREM1, TREM2, CSF-1 receptor,and any combination thereof, or of one or more cancer vaccines; (r)inhibition of PLCy/PKC/calcium mobilization; (s) inhibition of PI3K/Akt,Ras/MAPK signaling; and (t) decreasing efficacy of one or morechemotherapy agents, optionally wherein the one or more of thechemotherapy agents are gemcitabine, capecitabine, anthracyclines,doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel(Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide(Cytoxan®), carboplatin (Paraplatin®), and any combination thereof. Insome embodiments that may be combined with any of the precedingembodiments, the disease, disorder, or injury is cancer, and the agentexhibits one or more activities selected from the group consisting ofconsisting of: (a) increasing the number of tumor infiltrating CD3⁺ Tcells; (b) decreasing cellular levels of Siglec-9 in non-tumorigenicCD14+ myeloid cells, optionally wherein the non-tumorigenic CD14⁺myeloid cells are tumor infiltrating cells or optionally wherein thenon-tumorigenic CD14⁺ myeloid cells are present in blood; (c) reducingthe number of non-tumorigenic CD14⁺ myeloid cells, optionally whereinthe non-tumorigenic CD14⁺ myeloid cells are tumor infiltrating cells oroptionally wherein the non-tumorigenic CD14⁺ myeloid cells are presentin blood; (d) reducing PD-L1 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (e) reducing PD-L2 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (f) reducing B7-H2 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (g) reducingB7-H3 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (h)reducing CD200R levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (i) reducing CD163 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (j) reducing CD206 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (k) decreasing tumor growthrate of solid tumors; (l) reducing tumor volume; (m) increasing efficacyof one or more PD-1 inhibitors; (n) increasing efficacy of one or morecheckpoint inhibitor therapies and/or immune-modulating therapies,optionally wherein the one or more checkpoint inhibitor therapies and/orimmune-modulating therapies target one or more of CTLA4, the adenosinepathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; (o)increasing efficacy of one or more chemotherapy agents, optionallywherein the one or more of the chemotherapy agents are gemcitabine,capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin(Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®),5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin(Paraplatin®), and any combination thereof; (p) increasing proliferationof T cells in the presence of non-tumorigenic myeloid-derived suppressorcells (MDSC); and (q) inhibiting differentiation, survival, and/or oneor more functions of non-tumorigenic myeloid-derived suppressor cells(MDSC); and (r) killing Siglec-9-expressing immunosuppressor myeloidcells and/or CD14-expressing cells in solid tumors and associated bloodvessels when conjugated to a chemical or radioactive toxin. In someembodiments that may be combined with any of the preceding embodiments,the agent exhibits one or more activities selected from the groupconsisting of consisting of: (a) increasing the number of tumorinfiltrating CD3⁺ T cells; (b) decreasing cellular levels of CD33 innon-tumorigenic CD14+ myeloid cells, optionally wherein thenon-tumorigenic CD14+ myeloid cells are tumor infiltrating cells oroptionally wherein the non-tumorigenic CD14 myeloid cells are present inblood; (c) reducing the number of non-tumorigenic CD14+ myeloid cells,optionally wherein the non-tumorigenic CD14⁺ myeloid cells are tumorinfiltrating cells or optionally wherein the non-tumorigenic CD14⁺myeloid cells are present in blood; (d) reducing PD-L1 levels in one ormore cells, optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (e) reducing PD-L2 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (f) reducingB7-H2 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (g)reducing B7-H3 levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (h) reducing CD200R levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (i) reducing CD163 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (j) reducing CD206 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (k) decreasingtumor growth rate of solid tumors; (l) reducing tumor volume; (m)increasing efficacy of one or more PD-1 inhibitors; (n) increasingefficacy of one or more checkpoint inhibitor therapies and/orimmune-modulating therapies, optionally wherein the one or morecheckpoint inhibitor therapies and/or immune-modulating therapies targetone or more of CTLA4, the adenosine pathway, PD-L1, PD-L2, OX40, TIM3,LAG3, or any combination thereof; (o) increasing efficacy of one or morechemotherapy agents, optionally wherein the one or more of thechemotherapy agents are gemcitabine, capecitabine, anthracyclines,doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel(Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide(Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (p)increasing proliferation of T cells in the presence of non-tumorigenicmyeloid-derived suppressor cells (MDSC); and (q) inhibitingdifferentiation, survival, and/or one or more functions ofnon-tumorigenic myeloid-derived suppressor cells (MDSC); and (r) killingCD33-expressing immunosuppressor myeloid cells and/or CD14-expressingcells in solid tumors and associated blood vessels when conjugated to achemical or radioactive toxin. In some embodiments, the cancer isselected from the group consisting of bladder cancer, brain cancer,breast cancer, colon cancer, rectal cancer, endometrial cancer, kidneycancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer,melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer,ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyeloid leukemia (CML), and multiple myeloma. In some embodiments, theagent is selected from the group consisting of an antibody, a solubleSiglec-9 receptor, a Siglec-9-Fc fusion protein, a Siglec-9immunoadhesin, an antisense molecule, an siRNA, a small moleculeinhibitor, a protein, and a peptide. In some embodiments, the agent isan isolated anti-Siglec-9 antibody. In some embodiments, theanti-Siglec-9 antibody is the anti-Siglec-9 antibody of any of thepreceding embodiments. In some embodiments, the one or more immune cellsare selected from the group consisting of dendritic cells, macrophages,neutrophils, NK cells, microglia, T cells, T helper cells, cytotoxic Tcells, and any combination thereof.

Other aspects of the present disclosure relate to a method of inducingor promoting the survival, maturation, functionality, migration, orproliferation of one or more immune cells in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of an agent that decreases cellular levels of Siglec-9,inhibits interaction between Siglec-9 and one or more Siglec-9 ligands,or both. Other aspects of the present disclosure relate to an agent thatdecreases cellular levels of Siglec-9, inhibits interaction betweenSiglec-9 and one or more Siglec-9 ligands, or both for use in inducingor promoting the survival, maturation, functionality, migration, orproliferation of one or more immune cells in an individual in needthereof. Other aspects of the present disclosure relate to use of anagent that decreases cellular levels of Siglec-9, inhibits interactionbetween Siglec-9 and one or more Siglec-9 ligands, or both in themanufacture of a medicament for inducing or promoting the survival,maturation, functionality, migration, or proliferation of one or moreimmune cells in an individual in need thereof. In some embodiments theone or more immune cells are selected from the group consisting ofdendritic cells, macrophages, neutrophils, NK cells, microglia, T cells,T helper cells, cytotoxic T cells, and any combination thereof. In someembodiments, the agent is selected from the group consisting of anantibody, a soluble Siglec-9 receptor, a Siglec-9-Fc fusion protein, aSiglec-9 immunoadhesin, an antisense molecule, an siRNA, a smallmolecule inhibitor, a protein, and a peptide. In some embodiments, theagent is an isolated anti-Siglec-9 antibody. In some embodiments, theanti-Siglec-9 antibody is the anti-Siglec-9 antibody of any of thepreceding embodiments. In some embodiments, the one or more immune cellsare selected from the group consisting of dendritic cells, macrophages,neutrophils, NK cells, microglia, T cells, T helper cells, cytotoxic Tcells, and any combination thereof.

Other aspects of the present disclosure relate a method of decreasingthe activity, functionality, or survival of regulatory T cells,tumor-imbedded immunosuppressor dendritic cells, tumor-imbeddedimmunosuppressor macrophages, myeloid-derived suppressor cells,tumor-associated macrophages, acute myeloid leukemia (AML) cells,chronic lymphocytic leukemia (CLL) cell, or chronic myeloid leukemia(CML) cells in an individual in need thereof, comprising administeringto the individual a therapeutically effective amount of an agent thatbinds or interacts with Siglec-9. Other aspects of the presentdisclosure relate to use of an agent that binds or interacts withSiglec-9 for use in decreasing the activity, functionality, or survivalof regulatory T cells, tumor-imbedded immunosuppressor dendritic cells,tumor-imbedded immunosuppressor macrophages, myeloid-derived suppressorcells, tumor-associated macrophages, acute myeloid leukemia (AML) cells,chronic lymphocytic leukemia (CLL) cell, or chronic myeloid leukemia(CML) cells in an individual in need thereof. Other aspects of thepresent disclosure relate to use of an agent that binds or interactswith Siglec-9 in the manufacture of a medicament for decreasing theactivity, functionality, or survival of regulatory T cells,tumor-imbedded immunosuppressor dendritic cells, tumor-imbeddedimmunosuppressor macrophages, myeloid-derived suppressor cells,tumor-associated macrophages, acute myeloid leukemia (AML) cells,chronic lymphocytic leukemia (CLL) cell, or chronic myeloid leukemia(CML) cells in an individual in need thereof. In some embodiments, theagent is an isolated anti-Siglec-9 antibody or anti-Siglec-9 antibodyconjugate. In some embodiments, the anti-Siglec-9 antibody is theanti-Siglec-9 antibody of any of the preceding embodiments.

Other aspects of the present disclosure relate a method of decreasingcellular levels of Siglec-9 on one or more cells in an individual inneed thereof, comprising administering to the individual atherapeutically effective amount of an isolated anti-Siglec-9 antibody.Other aspects of the present disclosure relate to use of an isolatedanti-Siglec-9 antibody for use in decreasing cellular levels of Siglec-9on one or more cells in an individual in need thereof. Other aspects ofthe present disclosure relate to use of an isolated anti-Siglec-9antibody in the manufacture of a medicament for decreasing cellularlevels of Siglec-9 on one or more cells in an individual in needthereof. In some embodiments, the anti-Siglec-9 antibody decreasescellular levels of Siglec-9 in vivo. In some embodiments, theanti-Siglec-9 antibody is the anti-Siglec-9 antibody of any of thepreceding embodiments.

In some embodiments that may be combined with any of the precedingembodiments, the method further comprising administering to theindividual at least one antibody that specifically binds to aninhibitory checkpoint molecule, and/or one or more standard orinvestigational anti-cancer therapies. In some embodiments that may becombined with any of the preceding embodiments, the at least oneantibody that specifically binds to an inhibitory checkpoint molecule isadministered in combination with the anti-Siglec-9 antibody. In someembodiments that may be combined with any of the preceding embodiments,the at least one antibody that specifically binds to an inhibitorycheckpoint molecule is selected from the group consisting of ananti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, ananti-PD-1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, andanti-HVEM antibody, an anti-B- and T-lymphocyte attenuator (BTLA)antibody, an anti-Killer inhibitory receptor (KIR) antibody, ananti-GAL9 antibody, an anti-TIM-1 antibody, an anti-TIM3 antibody, ananti-TIM-4 antibody, an anti-A2AR antibody, an anti-CD39 antibody, ananti-CD73 antibody, an anti-LAG-3 antibody, an anti-phosphatidylserineantibody, an anti-CD27 antibody, an anti-CD30 antibody, an anti-TNFaantibody, an anti-CD33 antibody, an anti-Siglec-5 antibody, ananti-Siglec-7 antibody, an anti-Siglec-11 antibody, an antagonisticanti-TREM1 antibody, an antagonistic anti-TREM2 antibody, an anti-TIGITantibody, an anti-VISTA antibody, an anti-CD2 antibody, an anti-CD5antibody, and any combination thereof. In some embodiments that may becombined with any of the preceding embodiments, the one or more standardor investigational anti-cancer therapies are selected from the groupconsisting of radiotherapy, cytotoxic chemotherapy, targeted therapy,imatinib therapy, trastuzumab therapy, etanercept therapy, adoptive celltransfer (ACT) therapy, chimeric antigen receptor T cell transfer(CAR-T) therapy, vaccine therapy, and cytokine therapy. In someembodiments that may be combined with any of the preceding embodiments,the method further comprising administering to the individual at leastone antibody that specifically binds to an inhibitory cytokine. In someembodiments that may be combined with any of the preceding embodiments,the at least one antibody that specifically binds to an inhibitorycytokine is administered in combination with the anti-Siglec-9 antibody.In some embodiments that may be combined with any of the precedingembodiments, the at least one antibody that specifically binds to aninhibitory cytokine is selected from the group consisting of ananti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, andany combination thereof. In some embodiments that may be combined withany of the preceding embodiments, the method further comprisingadministering to the individual at least one agonistic antibody thatspecifically binds to a stimulatory checkpoint protein. In someembodiments that may be combined with any of the preceding embodiments,the at least one agonistic antibody that specifically binds to astimulatory checkpoint protein is administered in combination with theanti-Siglec-9 antibody. In some embodiments that may be combined withany of the preceding embodiments, the at least one agonistic antibodythat specifically binds to a stimulatory checkpoint protein is selectedfrom the group consisting of an agonist anti-CD40 antibody, an agonistanti-OX40 antibody, an agonist anti-ICOS antibody, an agonist anti-CD28antibody, an agonistic anti-TREM1 antibody, an agonistic anti-TREM2antibody, an agonist anti-CD137/4-1BB antibody, an agonist anti-CD27antibody, an agonist anti-glucocorticoid-induced TNFR-related proteinGITR antibody, an agonist anti-CD30 antibody, an agonist anti-BTLAantibody, an agonist anti-HVEM antibody, an agonist anti-CD2 antibody,an agonist anti-CD5 antibody, and any combination thereof. In someembodiments that may be combined with any of the preceding embodiments,the method further comprising administering to the individual at leastone stimulatory cytokine. In some embodiments that may be combined withany of the preceding embodiments, the at least one stimulatory cytokineis administered in combination with the anti-Siglec-9 antibody. In someembodiments that may be combined with any of the preceding embodiments,the at least one stimulatory cytokine is selected from the groupconsisting of IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, IL-20 familymembers, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-17, IL-18,IL-23, CXCL10, IL-33, MCP-1, MIP-1-beta, and any combination thereof.

Other aspects of the present disclosure relate to a method of assessingresponsiveness of a subject in need thereof to an agent that binds orinteracts with Siglec-9, the method comprising: a. measuring theexpression levels of CD45⁺ and CD14⁺ on non-tumorigenic myeloid cells ina blood sample obtained from the subject prior to administering to thesubject an anti-Siglec-9 antibody; b. administering to the subject atherapeutically effective amount of the agent; and c. measuring theexpression levels of CD45⁺ and CD14⁺ on non-tumorigenic myeloid cells ina blood sample obtained from the subject after administration of theanti-Siglec-9 antibody, wherein a reduction in the levels of CD45⁺CD14⁺on non-tumorigenic myeloid cells after administration of theanti-Siglec-9 antibody indicates the subject is responsive to the agent.In some embodiments, the method of assessing responsiveness furthercomprises administering one or more additional therapeutically effectiveamounts of the agent. In some embodiments that may be combined with anyof the preceding embodiments, the agent is selected from the groupconsisting of an antibody, a soluble Siglec-9 receptor, a Siglec-9-Fcfusion protein, a Siglec-9 immunoadhesin, a soluble Siglec receptor, aSiglec-Fc fusion protein, a Siglec immunoadhesin, an antisense molecule,an siRNA, a small molecule inhibitor, a protein, and a peptide. In someembodiments that may be combined with any of the preceding embodiments,the agent is an isolated anti-Siglec-9 antibody or anti-Siglec-9antibody conjugate. In some embodiments that may be combined with any ofthe preceding embodiments, the anti-Siglec-9 antibody is theanti-Siglec-9 antibody of any of the preceding embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an amino acid sequence alignment between human Siglec-9(SEQ ID NO: 1), chimpanzee Siglec-9 (SEQ ID NO: 2), green monkeySiglec-9 (SEQ ID NO: 3), rhesus macaque Siglec-9 (SEQ ID NO: 4), andmouse Siglec-9 (SEQ ID NO: 5). An asterisk (“*”) indicates positionswhich have a single, fully conserved residue; A colon (“:”) indicatesconservation between groups of strongly similar properties—scoring >0.5in the Gonnet PAM 250 matrix; and a period (“.”) indicates conservationbetween groups of weakly similar properties—scoring=<0.5 in the GonnetPAM 250 matrix. FIG. 1B depicts binding reactivity in percentage towild-type Siglec-9 (% WT) of antibodies of the present disclosure to theindicated Siglec-9 mutants. FIG. 1C depicts a Phyre2-derived model ofSiglec-9 (based on the crystal structure of Siglec-5; PDB ID 2ZG2)indicating amino acid residues involved in binding of anti-Siglec-9antibody 2D4. Critical residues for binding are indicated with redspheres. FIG. 1D depicts a Phyre2-derived model of Siglec-9 (based onthe crystal structure of Siglec-5; PDB ID 2ZG2) indicating amino acidresidues involved in binding of anti-Siglec-9 antibody 5C6. Criticalresidues for binding are indicated with red spheres. FIG. 1E depicts aPhyre2-derived model of Siglec-9 (based on the crystal structure ofSiglec-5; PDB ID 2ZG2) indicating amino acid residues involved inbinding of anti-Siglec-9 antibody 12B 12. Critical residues for bindingare indicated with red spheres.

FIG. 2 shows glycan-binding specificities of human Siglec proteins, suchas Siglec-9. This figure shows a summary of the most commonly reportedspecificities for the most commonly studied sialylated glycans. Relativebinding within studies of each Siglec is indicated as ++, strongbinding; +, detectable binding; and −, very weak or undetectablebinding. Not shown is the recently reported strong-binding preference ofhSiglec-8 and mSiglec-F for 6′-sulfated-sialyl-Lewis x (sLex) and ofhSiglec-9 for 6-sulfated-sLex. With a few exceptions (CD22 and MAG),results of binding specificity studies of human Siglecs by differentinvestigators using different assays have varied significantly. Inaddition to assay formats and glycan linker issues, the density andarrangement of the ligands studied could be responsible for thisvariation (Varki et al., (2006) Glycobiol. 16:1R-27R).

FIG. 3 shows the structure and metabolism of disialogangliosides inmammalian brain. The nomenclature of disialogangliosides in the figurefollows the system of Svennerholm (1964) J. Lipid Res. 5:145-155 (ArigaT et al. (2008) J. Lipid Res. 49:1157-1175).

FIG. 4 depicts results of FACS analysis demonstrating Siglec-9expression in human primary immune cells.

FIG. 5 depicts FACS analysis of Siglec-9 antibodies binding to humanprimary dendritic cells compared to isotype controls.

FIG. 6A depicts Biacore sensorgrams showing binding affinity of Siglec-9antibodies of the present disclosure to purified Siglec-9-his taggedprotein. FIG. 6B depicts ForteBio sensorgrams showing binding affinityof Siglec-9 antibodies of the present disclosure to purifiedSiglec-9-his tagged protein. FIG. 6C depicts shows a schematic forcombining antibody light chain variable region (VL) sequences ofhumanized versions of anti-Siglec-9 antibody 5C6 (AbM S9-5C6.3).Additional variations are listed below each sequence. The figureincludes sequences for versions of humanized antibody 5C6. In thefigure, IGKV230*01 (SEQ ID NO: 213); Joining region (SEQ ID NO: 214);5C6.3 (SEQ ID NO: 215); 2-30*01 (SEQ ID NO: 216); h5C6.3-L1 (SEQ ID NO:217); h5C6.3-L2 (SEQ ID NO: 218); h5C6.3-L3 (SEQ ID NO: 219). FIG. 6Dshows a schematic for combining antibody heavy chain variable region(VH) sequences of humanized versions of anti-Siglec-9 antibody 5C6 (AbMS9-5C6.3). Additional variations are listed below each sequence. Thefigure includes sequences for versions of humanized antibody 5C6. In thefigure, IGHV1-18*01 (SEQ ID NO: 220); Joining region (SEQ ID NO: 221);5C6.3 (SEQ ID NO: 222); 1-18*01 (SEQ ID NO: 223); h5C6.3-H1 (SEQ ID NO:224); h5C6.3-H2 (SEQ ID NO: 225); h5C6.3-H3 (SEQ ID NO: 226); h5C6.3-H4(SEQ ID NO: 227). FIG. 6E shows a schematic for combining antibody lightchain variable region (VL) sequences of humanized versions ofanti-Siglec-9 antibody 12B12 (AbM S9-12B12.2). Additional variations arelisted below each sequence. The figure includes sequences for versionsof humanized antibody 12B12. In the figure, IGKV1-39*01 (SEQ ID NO:228); Joining region (SEQ ID NO: 229); 12B12.2 (SEQ ID NO: 230); 1-39*01(SEQ ID NO: 231); h12B12.2-L1 (SEQ ID NO: 232); h12B12.2-L2 (SEQ ID NO:233); h12B12.2-L3 (SEQ ID NO: 234). FIG. 6F shows a schematic forcombining antibody heavy chain variable region (VH) sequences ofhumanized versions of anti-Siglec-9 antibody 12B12 (AbM S9-12B 12.2).Additional variations are listed below each sequence. The figureincludes sequences for versions of humanized antibody 12B12. In thefigure, IGHV3-23*04 (SEQ ID NO: 235); Joining region (SEQ ID NO: 236);12B 12.2 (SEQ ID NO: 237); 3-23*04 (SEQ ID NO: 238); h12B12.2-H1 (SEQ IDNO: 239); h12B12.2-H2 (SEQ ID NO: 240). For FIG. 6C-6F, CDR sequencesare depicted in bold; residue numbering shown as sequential (seq) oraccording to Chothia; “b” refers to buried sidechain; “p” refers topartially buried; “i” refers to sidechain at interface between VH and VLdomains; sequence differences between human and murine germlines arenoted by an asterisk (*); potential additional mutations in frameworksare noted below the sequence; potential changes in CDR sequences arenoted below each CDR sequence (such changes may prevent asparagine (N)deamidation, tryptophan (W) oxidation, or isoaspartate (DG) formation).

FIG. 7A-7H depict Siglec-9 antibody-dependent downregulation of cellsurface Siglec-9 receptor on various human cell types. FIG. 7A depictsTHP-1 acute monocytic lymphoma cells. FIG. 7B depicts human primarymonocytes. FIG. 7C depicts human primary microglia. FIG. 7D depictshuman primary macrophages. FIG. 7E depicts human primary dendriticcells. FIG. 7F depicts antibody titration with human primary dendriticcells. FIG. 7G shows in vivo reduction in cell surface levels ofSiglec-9 following antibody treatment in vivo. FIG. 7H shows expressionof unrelated receptor CD33. CD33 was used as a control. Cell surfacelevels of CD33 were not significantly reduced following antibodytreatment in vivo. FIG. 7I depicts an antibody concentration titrationcurve for reducing cell surface expression of Siglec-9 on human primarymacrophages with Siglec-9 antibodies 5C6 (S9-5C6), 12B12 (S9-12B 12),17C2 (S9-17C2), and isotype control (mIgG2a). FIG. 7J depicts anantibody concentration titration curve for reducing cell surfaceexpression of control receptor CD11b on human primary macrophages withSiglec-9 antibodies 5C6 (S9-5C6), 12B12 (S9-12B12), 17C2 (S9-17C2), andisotype control (mIgG2a). FIG. 7K depicts Siglec-9 and CD33 expressionin peripheral myeloid cells from humanized NSG mice 21 days aftertreatment with anti-Siglec-9 antibody 2D4 or isotype control antibody(MOPC-21). FIG. 7L shows in vivo reduction in cell surface levels ofSiglec-9 following antibody treatment in vivo. FIG. 7M shows expressionof unrelated receptor CD33. CD33 was used as a control. Cell surfacelevels of CD33 were not significantly reduced following antibodytreatment in vivo. FIG. 7N shows FACS gating strategy for blood samplesfrom humanized NSG mice 21 days after treatment with anti-Siglec-9antibody 2D4 or isotype control antibody (MOPC21). FIG. 7O depictslevels of Siglec-9 surface expression and TREM2 surface expression onhuman dendritic cells after treatment with an anti-Siglec-9 antibody 2D4or an isotype control antibody, as compared to a no antibody control.FIG. 7P depicts levels of CD11c surface expression, Siglec-9 surfaceexpression, and TREM2 surface expression on human dendritic cells aftertreatment with an anti-Siglec-9 antibody 2D4. FIG. 7Q depicts ability ofSiglec-9 antibodies of the present disclosure to block binding ofSiglec-9 (Siglec-9-FC) to sialic acid ligands endogenously expressed onU937 cancer cells.

FIG. 8 depicts FACS analysis showing that sialic acid ligands ondendritic cells restrict T cell proliferation during mixed lymphocytereaction with human primary cells.

FIG. 9 depicts results showing that sialic acid Siglec-9 ligands ondendritic cells restrict T cell proliferation during mixed lymphocytereaction.

FIG. 10A-10H depict results showing Siglec-9 receptor and increasedSiglec-9 ligand expression on human myeloid cells induced by variousstimuli. FIGS. 10A and 10B depict results showing Siglec-9 receptor andincreased Siglec-9 ligand expression on human primary dendritic cellsafter treatment with tumor supernatant. FIGS. 10C and 10D depict resultsshowing increased Siglec-9 ligand expression on human primary dendriticcells after treatment with tumor supernatant. FIGS. 10E and 10F depictresults showing Siglec-9 receptor expression on human dendritic cellsduring LPS-induced inflammation. FIGS. 10G and 10H depict resultsshowing an increase in sialic acid expression on human myeloid cellsduring LPS-induced inflammation. These finding indicate that tumors canevade immune surveillance by upregulating the level of inhibitoryligands to Siglec-9 on the tumor cells and on immune cells.

FIG. 11 depicts results showing that sialidase treatment to removeSiglec-9 ligands from E. coli increases phagocytosis by human primarydendritic cells.

FIG. 12 depicts Siglec-9 ligand expression in brain sections from anAlzheimer's disease brain (AD) and a healthy brain (non-AD). FIG. 12shows immunohistochemistry staining of Siglec-9-Fc in AD and non-ADbrain samples from two donors (Donor 1 and Donor 2).

FIG. 13 depicts results showing that expression of inhibitory Siglec-9ligands is increased in lung tumor cells, melanoma cells, and coloncancer cells. The results indicate that inhibitory Siglec-9 ligandscontribute to cancer pathology in these tumor types.

FIG. 14 depicts expression of Siglec-9 in cells of NOG mice injectedwith human peripheral blood cells. 12 weeks after engraftment with humanfetal liver CD34⁺ cells, humanized mice were transplanted withpatient-derived melanoma. Peripheral blood, spleen, and tumor tissueswere dissociated and analyzed for immune cell markers and Siglec-9expression. For FACS analysis, human hematopoietic cells were identifiedby CD45 expression, then gated CD14+, CD3+ populations. The resultsindicate that inhibitory Siglec-9 ligands contribute to cancer pathologyin this tumor type.

FIG. 15A depicts a PD-1/Siglec-9 combination antibody treatment protocolfor a mouse model of patient-derived cancer in immunologically humanizedmice. FIG. 15B depicts in vivo reduction in cell surface levels ofSiglec-9 on CD14+ myeloid cells from blood samples of mice treated withKeytruda® (pembrolizumab) anti-PD-1 antibody alone or in combinationwith anti-Siglec-9 antibody 2D4. FIG. 15C depicts in vivo cell surfacelevels of control receptor CD33 on CD14+ myeloid cells from bloodsamples of mice treated with Keytruda® (pembrolizumab) anti-PD-1antibody alone or in combination with anti-Siglec-9 antibody 2D4. FIG.15D depicts in vivo reduction in peripheral blood CD14⁺ myeloid cellsfrom mice treated with Keytruda® (pembrolizumab) anti-PD-1 antibodyalone or in combination with anti-Siglec-9 antibody 2D4. FIG. 15Edepicts in vivo increase in peripheral blood CD3⁺ T cells in bloodsamples from mice treated with Keytruda® (pembrolizumab) anti-PD-1antibody alone or in combination with anti-Siglec-9 antibody 2D4. FIG.15F depicts in vivo reduction in tumor infiltrating CD14⁺ myeloid cellsfrom mice treated with Keytruda® (pembrolizumab) anti-PD-1 antibodyalone or in combination with anti-Siglec-9 antibody 2D4. FIG. 15Gdepicts in vivo increase in tumor infiltrating CD3⁺ T cells from micetreated with Keytruda® (pembrolizumab) anti-PD-1 antibody alone or incombination with anti-Siglec-9 antibody 2D4. FIG. 15H depicts mean tumorvolume after treatment with Keytruda® (pembrolizumab) anti-PD-1 antibodyalone or in combination with anti-Siglec-9 antibody 2D4 in mice thatwere engrafted with human immune stem cells from human donor 984480112.FIG. 15I depicts mean tumor volume after treatment with Keytruda®(pembrolizumab) anti-PD-1 antibody alone or in combination withanti-Siglec-9 antibody 2D4 in mice that were engrafted with human immunestem cells from human donor 17509112. FIG. 15J depicts mean tumor volumeafter treatment with Keytruda® (pembrolizumab) anti-PD-1 antibody aloneor in combination with anti-Siglec-9 antibody 2D4 in mice that wereengrafted with human immune stem cells from human donor 165547112.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993).

Definitions

As used herein, the term “preventing” includes providing prophylaxiswith respect to occurrence or recurrence of a particular disease,disorder, or condition in an individual. An individual may bepredisposed to, susceptible to a particular disease, disorder, orcondition, or at risk of developing such a disease, disorder, orcondition, but has not yet been diagnosed with the disease, disorder, orcondition.

As used herein, an individual “at risk” of developing a particulardisease, disorder, or condition may or may not have detectable diseaseor symptoms of disease, and may or may not have displayed detectabledisease or symptoms of disease prior to the treatment methods describedherein. “At risk” denotes that an individual has one or more riskfactors, which are measurable parameters that correlate with developmentof a particular disease, disorder, or condition, as known in the art. Anindividual having one or more of these risk factors has a higherprobability of developing a particular disease, disorder, or conditionthan an individual without one or more of these risk factors.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual being treatedduring the course of clinical pathology. Desirable effects of treatmentinclude decreasing the rate of progression, ameliorating or palliatingthe pathological state, and remission or improved prognosis of aparticular disease, disorder, or condition. An individual issuccessfully “treated”, for example, if one or more symptoms associatedwith a particular disease, disorder, or condition are mitigated oreliminated.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. An effective amount can be provided in one or moreadministrations. An effective amount herein may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the treatment to elicit a desiredresponse in the individual. An effective amount is also one in which anytoxic or detrimental effects of the treatment are outweighed by thetherapeutically beneficial effects. For prophylactic use, beneficial ordesired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asdecreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, enhancingeffect of another medication such as via targeting, delaying theprogression of the disease, and/or prolonging survival. An effectiveamount of drug, compound, or pharmaceutical composition is an amountsufficient to accomplish prophylactic or therapeutic treatment eitherdirectly or indirectly. As is understood in the clinical context, aneffective amount of a drug, compound, or pharmaceutical composition mayor may not be achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective amount” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disease, disorder, or condition. A therapeutically effectiveamount herein may vary according to factors such as the disease state,age, sex, and weight of the patient, and the ability of the Siglec-9protein antagonist to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the Siglec-9 protein antagonist are outweighed bythe therapeutically beneficial effects.

As used herein, administration “in conjunction” with another compound orcomposition includes simultaneous administration and/or administrationat different times. Administration in conjunction also encompassesadministration as a co-formulation or administration as separatecompositions, including at different dosing frequencies or intervals,and using the same route of administration or different routes ofadministration.

An “individual” for purposes of treatment, prevention, or reduction ofrisk refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sport, or pet animals, such as dogs,horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats,cats, and the like. Preferably, the individual is human.

The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. The pairing of a V_(H) and V_(L) together forms a singleantigen-binding site. For the structure and properties of the differentclasses of antibodies, see, e.g., Basic and Clinical Immunology, 8thEd., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (“κ”) and lambda (“λ”), based onthe amino acid sequences of their constant domains. Depending on theamino acid sequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated alpha (“α”), delta (“δ”), epsilon (“ε”), gamma(“γ”) and mu (“μ”), respectively. The γ and α classes are furtherdivided into subclasses (isotypes) on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thesubunit structures and three dimensional configurations of differentclasses of immunoglobulins are well known and described generally in,for example, Abbas et al., Cellular and Molecular Immunology, 4^(th) ed.(W.B. Saunders Co., 2000).

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

An “isolated” antibody, such as an anti-Siglec-9 antibody of the presentdisclosure, is one that has been identified, separated and/or recoveredfrom a component of its production environment (e.g., naturally orrecombinantly). Preferably, the isolated polypeptide is free ofassociation with all other contaminant components from its productionenvironment. Contaminant components from its production environment,such as those resulting from recombinant transfected cells, arematerials that would typically interfere with research, diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or non-proteinaceous solutes. In preferredembodiments, the polypeptide will be purified: (1) to greater than 95%by weight of antibody as determined by, for example, the Lowry method,and in some embodiments, to greater than 99% by weight; (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant T cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, an isolated polypeptide or antibody will beprepared by at least one purification step.

The “variable region” or “variable domain” of an antibody, such as ananti-Siglec-9 antibody of the present disclosure, refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “V_(H)” and “V_(L)”, respectively. These domains are generally themost variable parts of the antibody (relative to other antibodies of thesame class) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies, suchas anti-Siglec-9 antibodies of the present disclosure. The V domainmediates antigen binding and defines the specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the entire span of the variable domains.Instead, it is concentrated in three segments called hypervariableregions (HVRs) both in the light-chain and the heavy chain variabledomains. The more highly conserved portions of variable domains arecalled the framework regions (FR). The variable domains of native heavyand light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent-cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibody,such as an anti-Siglec-9 antibody of the present disclosure, obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations and/or post-translationmodifications (e.g., isomerizations, amidations) that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst one or more antigenic sites. In some embodiments, a monoclonalantibody of the present disclosure can be a bispecific antibody. Incontrast to polyclonal antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theone or more antigenic sites. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent disclosure may be made by a variety of techniques, including,for example, phage-display technologies (see, e.g., Clackson et al.,Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al.,J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat'l Acad. Sci.USA 101(34):12467-472 (2004); and Lee et al., J. Immunol. Methods284(1-2):119-132 (2004), the hybridoma method (e.g., Kohler andMilstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),and technologies for producing human or human-like antibodies in animalsthat have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Nat'lAcad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild etal., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol.14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93(1995).

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody, such as ananti-Siglec-9 antibody of the present disclosure, in its substantiallyintact form, as opposed to an antibody fragment. Specifically wholeantibodies include those with heavy and light chains including an Fcregion. The constant domains may be native sequence constant domains(e.g., human native sequence constant domains) or amino acid sequencevariants thereof. In some cases, the intact antibody may have one ormore effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995));single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

Papain digestion of antibodies, such as anti-Siglec-9 antibodies of thepresent disclosure, produces two identical antigen-binding fragments,called “Fab” fragments, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. The Fab fragment consistsof an entire L chain along with the variable region domain of the Hchain (V_(H)), and the first constant domain of one heavy chain(C_(H)1). Each Fab fragment is monovalent with respect to antigenbinding, i.e., it has a single antigen-binding site. Pepsin treatment ofan antibody yields a single large F(ab′)₂ fragment which roughlycorresponds to two disulfide linked Fab fragments having differentantigen-binding activity and is still capable of cross-linking antigen.Fab′ fragments differ from Fab fragments by having a few additionalresidues at the carboxy terminus of the C_(H)1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of antibodies, such as anti-Siglec-9 antibodiesof the present disclosure, comprise a portion of an intact antibody,generally including the antigen binding or variable region of the intactantibody or the F region of an antibody which retains or has modifiedFcR binding capability. Examples of antibody fragments include linearantibody, single-chain antibody molecules and multispecific antibodiesformed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48(1993).

As used herein, a “chimeric antibody” refers to an antibody(immunoglobulin), such as an anti-Siglec-9 antibody of the presentdisclosure, in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is(are) identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies ofinterest herein include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with an antigen ofinterest. As used herein, “humanized antibody” is used a subset of“chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies, such asanti-Siglec-9 antibodies of the present disclosure, are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR of therecipient are replaced by residues from an HVR of a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance, such as binding affinity. In general, a humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableloops correspond to those of a non-human immunoglobulin sequence, andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence, although the FR regions may include one or moreindividual FR residue substitutions that improve antibody performance,such as binding affinity, isomerization, immunogenicity, and the like.The number of these amino acid substitutions in the FR is typically nomore than 6 in the H chain, and in the L chain, no more than 3. Thehumanized antibody optionally will also comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one that possesses an amino-acid sequencecorresponding to that of an antibody, such as an anti-Siglec-9 antibodyof the present disclosure, produced by a human and/or has been madeusing any of the techniques for making human antibodies as disclosedherein. This definition of a human antibody specifically excludes ahumanized antibody comprising non-human antigen-binding residues. Humanantibodies can be produced using various techniques known in the art,including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Alsoavailable for the preparation of human monoclonal antibodies are methodsdescribed in Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991).See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74(2001). Human antibodies can be prepared by administering the antigen toa transgenic animal that has been modified to produce such antibodies inresponse to antigenic challenge, but whose endogenous loci have beendisabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181and 6,150,584 regarding XENOMOUSE™ technology). See also, for example,Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006) regardinghuman antibodies generated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody-variable domain, such as that of ananti-Siglec-9 antibody of the present disclosure, that are hypervariablein sequence and/or form structurally defined loops. Generally,antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three inthe VL (L1, L2, L3). In native antibodies, H3 and L3 display the mostdiversity of the six HVRs, and H3 in particular is believed to play aunique role in conferring fine specificity to antibodies. See, e.g., Xuet al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003)). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheHVRs that are EU or Kabat complementarity-determining regions (CDRs) arebased on sequence variability and are the most commonly used (Kabat etal., supra). Chothia refers instead to the location of the structuralloops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRsrepresent a compromise between the EU or Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbMantibody-modeling software. The “contact” HVRs are based on an analysisof the available complex crystal structures. The residues from each ofthese HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 1196-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65or 49-65 (a preferred embodiment) (H2), and 93-102, 94-102, or 95-102(H3) in the VH. The variable-domain residues are numbered according toEU or Kabat et al., supra, for each of these extended-HVR definitions.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

The phrase “variable-domain residue-numbering as in EU or Kabat” or“amino-acid-position numbering as in EU or Kabat,” and variationsthereof, refers to the numbering system used for heavy-chain variabledomains or light-chain variable domains of the compilation of antibodiesin EU or Kabat et al., supra. Using this numbering system, the actuallinear amino acid sequence may contain fewer or additional amino acidscorresponding to a shortening of, or insertion into, a FR or HVR of thevariable domain. For example, a heavy-chain variable domain may includea single amino acid insert (residue 52a according to Kabat) afterresidue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and82c, etc. according to Kabat) after heavy-chain FR residue 82. The EU orKabat numbering of residues may be determined for a given antibody byalignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence.

The EU or Kabat numbering system is generally used when referring to aresidue in the variable domain (approximately residues 1-107 of thelight chain and residues 1-113 of the heavy chain) (e.g., Kabat et al.,Sequences of Immunological Interest. 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The “EU or Kabatnumbering system” or “EU index” is generally used when referring to aresidue in an immunoglobulin heavy chain constant region (e.g., the EUindex reported in Kabat et al., supra). The “EU index as in Kabat”refers to the residue numbering of the human IgG1 EU antibody. Unlessstated otherwise herein, references to residue numbers in the variabledomain of antibodies means residue numbering by the Kabat numberingsystem. Unless stated otherwise herein, references to residue numbers inthe constant domain of antibodies means residue numbering by the EU orKabat numbering system (e.g., see United States Patent Publication No.2010-280227).

An “acceptor human framework” as used herein is a framework comprisingthe amino acid sequence of a VL or VH framework derived from a humanimmunoglobulin framework or a human consensus framework. An acceptorhuman framework “derived from” a human immunoglobulin framework or ahuman consensus framework may comprise the same amino acid sequencethereof, or it may contain pre-existing amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. Where pre-existing amino acid changes arepresent in a VH, preferable those changes occur at only three, two, orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may by 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework that represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Examples include for the VL, the subgroup may be subgroup kappaI, kappa II, kappa III or kappa IV as in Kabat et al., supra.Additionally, for the VH, the subgroup may be subgroup I, subgroup II,or subgroup III as in Kabat et al., supra.

An “amino-acid modification” at a specified position, e.g., of ananti-Siglec-9 antibody of the present disclosure, refers to thesubstitution or deletion of the specified residue, or the insertion ofat least one amino acid residue adjacent the specified residue.Insertion “adjacent” to a specified residue means insertion within oneto two residues thereof. The insertion may be N-terminal or C-terminalto the specified residue. The preferred amino acid modification hereinis a substitution.

An “affinity-matured” antibody, such as an anti-Siglec-9 antibody of thepresent disclosure, is one with one or more alterations in one or moreHVRs thereof that result in an improvement in the affinity of theantibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by VH- and VL-domain shuffling. Randommutagenesis of HVR and/or framework residues is described by, forexample: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As use herein, the term “specifically recognizes” or “specificallybinds” refers to measurable and reproducible interactions such asattraction or binding between a target and an antibody, such as ananti-Siglec-9 antibody of the present disclosure, that is determinativeof the presence of the target in the presence of a heterogeneouspopulation of molecules including biological molecules. For example, anantibody, such as an anti-Siglec-9 antibody of the present disclosure,that specifically or preferentially binds to a target or an epitope isan antibody that binds this target or epitope with greater affinity,avidity, more readily, and/or with greater duration than it binds toother targets or other epitopes of the target. It is also understood byreading this definition that, for example, an antibody (or a moiety)that specifically or preferentially binds to a first target may or maynot specifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. An antibody thatspecifically binds to a target may have an association constant of atleast about 10³ M⁻¹ or 10⁴M⁻¹, sometimes about 10⁵M⁻¹ or 10⁶ M⁻¹, inother instances about 10⁶M⁻¹ or 10⁷M⁻¹, about 10⁸ M⁻¹ to 10⁹ M⁻¹, orabout 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher. A variety of immunoassay formatscan be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

As used herein, an “interaction” between a Siglec-9 protein and a secondprotein encompasses, without limitation, protein-protein interaction, aphysical interaction, a chemical interaction, binding, covalent binding,and ionic binding. As used herein, an antibody “inhibits interaction”between two proteins when the antibody disrupts, reduces, or completelyeliminates an interaction between the two proteins. An antibody of thepresent disclosure, or fragment thereof, “inhibits interaction” betweentwo proteins when the antibody or fragment thereof binds to one of thetwo proteins.

An “agonist” antibody or an “activating” antibody is an antibody, suchas an agonist anti-Siglec-9 antibody of the present disclosure, thatinduces (e.g., increases) one or more activities or functions of theantigen after the antibody binds the antigen.

A “blocking” antibody, an “antagonist” antibody, or an “inhibitory”antibody is an antibody, such as an anti-Siglec-9 antibody of thepresent disclosure, that inhibits or reduces (e.g., decreases) antigenbinding to one or more ligand after the antibody binds the antigen,and/or that inhibits or reduces (e.g., decreases) one or more activitiesor functions of the antigen after the antibody binds the antigen. Insome embodiments, blocking antibodies, antagonist antibodies, orinhibitory antibodies substantially or completely inhibit antigenbinding to one or more ligand and/or one or more activities or functionsof the antigen.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU or Kabat numberingsystem) of the Fc region may be removed, for example, during productionor purification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the present disclosure include human IgG1,IgG2, IgG3 and IgG4.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (“ITAM”) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (“ITIM”) in its cytoplasmic domain. (see, e.g., M.Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126: 330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. FcRs can also increasethe serum half-life of antibodies.

Binding to FcRn in vivo and serum half-life of human FcRn high-affinitybinding polypeptides can be assayed, e.g., in transgenic mice ortransfected human cell lines expressing human FcRn, or in primates towhich the polypeptides having a variant Fc region are administered. WO2004/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al., J. Biol.Chem. 9(2):6591-6604 (2001).

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequencerefers to the percentage of amino acid residues in a candidate sequencethat are identical with the amino acid residues in the specific peptideor polypeptide sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms known in the art needed toachieve maximal alignment over the full length of the sequences beingcompared.

An “isolated” cell is a molecule or a cell that is identified andseparated from at least one contaminant cell with which it is ordinarilyassociated in the environment in which it was produced. In someembodiments, the isolated cell is free of association with allcomponents associated with the production environment. The isolated cellis in a form other than in the form or setting in which it is found innature. Isolated cells are distinguished from cells existing naturallyin tissues, organs, or individuals. In some embodiments, the isolatedcell is a host cell of the present disclosure.

An “isolated” nucleic acid molecule encoding an antibody, such as ananti-Siglec-9 antibody of the present disclosure, is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in theenvironment in which it was produced. Preferably, the isolated nucleicacid is free of association with all components associated with theproduction environment. The isolated nucleic acid molecules encoding thepolypeptides and antibodies herein is in a form other than in the formor setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from nucleic acid encoding thepolypeptides and antibodies herein existing naturally in cells.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of the present disclosure.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

As used herein, the term “apoptosis” refers to gene-directed process ofintracellular cell destruction. Apoptosis is distinct from necrosis; itincludes cytoskeletal disruption, cytoplasmic shrinkage andcondensation, expression of phosphatidylserine on the outer surface ofthe cell membrane and blebbing, resulting in the formation of cellmembrane bound vesicles or apoptotic bodies. The process is alsoreferred to as “programmed cell death.” During apoptosis, characteristicphenomena such as curved cell surfaces, condensation of nuclearchromatin, fragmentation of chromosomal DNA, and loss of mitochondrialfunction are observed. Various known technologies may be used to detectapoptosis, such as staining cells with Annexin V, propidium iodide, DNAfragmentation assay and YO-PRO-1 (Invitrogen). In some embodiments,staining with Annexin V and propidium iodide may be used, and thecombined percentages of the Annexin V+/PI+, Annexin V+/PI− and AnnexinV−/PI+ populations are considered as dead cells.

As used herein, the term “agent that decreases cellular levels ofSiglec-9, inhibits interaction between Siglec-9 and one or more Siglec-9ligands, or both” refers to a molecule that reduces (includingsignificantly), decreases, blocks, inhibits, or interferes with aSiglec-9 (mammalian, such as a human Siglec-9) biological activity invitro, in situ, and/or in vivo. The term “agent” implies no specificmechanism of biological action whatsoever, and expressly includes andencompasses all possible pharmacological, physiological, and biochemicalinteractions with a Siglec-9 whether direct or indirect, and whetherinteracting with a Siglec-9, one or more of its ligands, or throughanother mechanism, and its consequences which can be achieved by avariety of different, and chemically divergent, compositions. Exemplaryagents include, without limitation, an anti-Siglec-9 antibody thatspecifically binds to a Siglec-9, a soluble Siglec-9 receptor protein, asoluble Siglec-9-Fc fusion protein (e.g., Siglec-9 immunoadhesin), asoluble Siglec receptor that binds to a Siglec-9 ligand, a Siglec-Fcfusion protein (e.g., Siglec immunoadhesin) that binds to a Siglec-9ligand, an anti-sense molecule directed to a nucleic acid encoding aSiglec-9, a short interfering RNA (“siRNA”) molecule directed to anucleic acid encoding a Siglec-9, a Siglec-9 inhibitory compound, an RNAor DNA aptamer that binds to a Siglec-9, and a Siglec-9 structuralanalog. In some embodiments, a Siglec-9 inhibitor (e.g., an antibody)binds (physically interacts with) an agent that decreases cellularlevels of Siglec-9, inhibits interaction between Siglec-9 and one ormore Siglec-9 ligands, or both, binds to a Siglec-9 ligand, and/orinhibits (reduces) Siglec-9 synthesis or production. In otherembodiments, an agent of the present disclosure inhibitor binds aSiglec-9 and prevents its binding to one or more of its ligands. Instill other embodiments, an agent of the present disclosure reduces oreliminates expression (i.e., transcription or translation) of aSiglec-9. Examples of types of agent that decreases cellular levels ofSiglec-9, inhibits interaction between Siglec-9 and one or more Siglec-9ligands, or both are provided herein.

As used herein, the term “agent that binds or interacts with Siglec-9”refers to a molecule that either directly or indirectly interacts with aSiglec-9 protein. The term “agent” implies no specific mechanism ofbiological action whatsoever, and expressly includes and encompasses allpossible pharmacological, physiological, and biochemical interactionswith a Siglec-9 whether direct or indirect, and whether interacting witha Siglec-9 or through another mechanism, and its consequences which canbe achieved by a variety of different, and chemically divergent,compositions. Exemplary agents include, without limitation, ananti-Siglec-9 antibody that specifically binds to a Siglec-9.

As used herein, the term “RNA interference” or “RNAi” refers generallyto a process in which a double-stranded RNA molecule or a short hairpinRNA molecule reducing or inhibiting the expression of a nucleic acidsequence with which the double-stranded or short hairpin RNA moleculeshares substantial or total homology. The term “short interfering RNA”or “siRNA” or “RNAi agent” refers to an RNA sequence that elicits RNAinterference. See Kreutzer et al., WO 00/44895; Zernicka-Goetz et al.,WO 01/36646; Fire, WO 99/32619; Mello and Fire, WO 01/29058. As usedherein, siRNA molecules include RNA molecules encompassing chemicallymodified nucleotides and non-nucleotides. The term “ddRNAi agent” refersto a DNA-directed RNAi agent that is transcribed from an exogenousvector. The terms “short hairpin RNA” or “shRNA” refer to an RNAstructure having a duplex region and a loop region. In certainembodiments, ddRNAi agents are expressed initially as shRNAs.

As used herein, the term “aptamer” refers to a heterologousoligonucleotide capable of binding tightly and specifically to a desiredmolecular target, such as, for example, common metabolic cofactors(e.g., Coenzyme A, S-adenosyl methionine, and the like), proteins (e.g.,complement protein C5, antibodies, and the like), or conservedstructural elements in nucleic acid molecules (e.g., structuresimportant for binding of transcription factors and the like). Aptamerstypically comprise DNA or RNA nucleotide sequences ranging from about 10to about 100 nucleotides in length, from about 10 to about 75nucleotides in length, from about 10 to about 50 nucleotides in length,from about 10 to about 35 nucleotides in length, and from about 10 toabout 25 nucleotides in length. Synthetic DNA or RNA oligonucleotidescan be made using standard solid phase phosphoramidite methods andequipment, such as by using a 3900 High Throughput DNA Synthesizer™,available from Applied Biosystems (Foster City, Calif.). Aptamersfrequently incorporate derivatives or analogs of the commonly occurringnucleotides found in DNA and RNA (e.g., A, G, C, and T/U), includingbackbone or linkage modifications (e.g., peptide nucleic acid (PNA) orphosphothioate linkages) to increase resistance to nucleases, bindingavidity, or to otherwise alter their pharmacokinetic properties.Exemplary modifications are set forth in U.S. Pat. Nos. 6,455,308;4,469,863; 5,536,821; 5,541,306; 5,637,683; 5,637,684; 5,700,922;5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165; 5,929,226;5,977,296; 6,140,482; and in WIPO publications WO 00/56746 and WO01/14398. Methods for synthesizing oligonucleotides comprising suchanalogs or derivatives are disclosed, for example, in the patentpublications cited above, and in U.S. Pat. Nos. 6,455,308; 5,614,622;5,739,314; 5,955,599; 5,962,674; 6,117,992; and in WO 00/75372.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to an “antibody” is a reference tofrom one to many antibodies, such as molar amounts, and includesequivalents thereof known to those skilled in the art, and so forth.

It is understood that aspect and embodiments of the present disclosuredescribed herein include “comprising,” “consisting,” and “consistingessentially of” aspects and embodiments.

Overview

The present disclosure relates to agents (e.g., anti-Siglec-9antibodies) that decrease cellular levels of Siglec-9 and/or inhibitinteraction between Siglec-9 and one or more Siglec-9 ligands, or thatbind Siglec-9 without decreasing cellular levels of Siglec-9 and/orwithout inhibiting interaction between Siglec-9 and one or more Siglec-9ligands; methods of making and using such agents (e.g., anti-Siglec-9antibodies); pharmaceutical compositions containing such agents (e.g.,anti-Siglec-9 antibodies); nucleic acids encoding such agents (e.g.,anti-Siglec-9 antibodies); and host cells containing nucleic acidsencoding such agents (e.g., anti-Siglec-9 antibodies).

In some embodiments, anti-Siglec-9 antibodies of the present disclosurehave one or more antagonistic activities that are due, at least in part,to the ability of the antibodies inhibit the interaction betweenSiglec-9 and one or more natural glycan ligands. In some embodiments,the anti-Siglec-9 antibodies of the present disclosure may have one ormore antagonistic activities that are due, at least in part, to theability of the antibodies to reduce cellular expression (e.g., cellsurface expression) of Siglec-9 by inducing degradation, downregulation, cleavage, receptor desensitization, and/or lysosomaltargeting of Siglec-9.

In some embodiments, antibody-induced Siglec-9 activity can bedetermined or tested in vitro by any of the techniques disclosed herein(see, e.g., Examples 1-5), including, without limitation, testingplate-binding of full-length anti-Siglec-9 antibodies to increase thedensity of antibodies exposed to Siglec-9, cross-linking anti-Siglec-9antibodies with a secondary antibody, cross-linking anti-Siglec-9antibodies with cells that express one or more Fcg receptors (e.g.,FcgRIIB), using Siglec-9 antibodies in solution, and using Fab fragmentsof Siglec-9 antibodies.

Certain aspects of the present disclosure are based, at least in part,on the identification of agents, such as anti-Siglec-9 antibodies, thatexhibit the ability to compete with one or more Siglec-9 ligands forbinding to Siglec-9 and/or the ability to decrease cell surface levelsof Siglec-9 on cells, resulting in the reduction, neutralization,prevention, or curbing of one or more Siglec-9 activities. ExemplarySiglec-9 activities include, without limitation, phosphorylation ofTyr-433 and Tyr-456 by a Src family tyrosine kinase, such as Syk, LCK,FYM, and/or ZAP70; recruitment of and binding to the tyrosine-specificprotein phosphatases SHP1 and SHP2; recruitment of and binding toPLC-gamma1, which acts as a guanine nucleotide exchange factor forDynamini-1; recruitment of and binding to SH2-domain containing protein(e.g., Crk1); recruitment of and binding to the spleen tyrosine kinaseSyk; recruitment of and binding to SH3-SH2-SH3 growth factorreceptor-bound protein 2 (Grb2); recruitment of and binding to multipleSH2-containing proteins; modulated expression of one or morepro-inflammatory cytokines, such as FN-α4, IFN-beta, IL-113, IL-1alpha,TNF-α, IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ, OSM, CNTF,GM-CSF, IL-11, IL-12, IL-17, IL-18, CRP, MCP-1, and MIP-1-beta;modulated expression of one or more pro-inflammatory cytokines in one ormore cells selected from macrophages, neutrophils, NK cells, dendriticcells, hone marrow-derived dendritic cells, monocytes, osteoclasts, Tcells, T helper cells, cytotoxic T cells, granulocytes, and microglialcells; increased expression of one or more anti-inflammatory cytokines,such as IL-4, IL-10, IL-13, IL-35, IL-16, TGF-beta, IL-1Ra, G-CSF, andsoluble receptors for TNF, IFN-beta1a, IFN-beta1b, or IL-6; modulatedexpression of one or more anti-inflammatory cytokines in one or morecells selected from macrophages, neutrophils, NK cells, dendritic cells,bone marrow-derived dendritic cells, monocytes, osteoclasts, T cells, Thelper cells, cytotoxic T cells, granulocytes, and microglial cells;modulate expression of one or more proteins selected from C qa, C1qB,C1qC, C1s, C1R, C4, C2, C3, ITGB2, HMOX1, LAT2, CASP1, CSTA, VSIG4,MS4A4A, C3AR1, GPX1, TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX, andPYCARD; inhibition of extracellular signal-regulated kinase (ERK)phosphorylation; decreasing tyrosine phosphorylation on one or morecellular proteins, optionally, wherein the one or more cellular proteinscomprise ZAP-70 and the tyrosine phosphorylation occurs on Tyr-319 ofZAP-70; modulated expression of C—C chemokine receptor 7 (CCR7);inhibition of microglial cell chemotaxis toward CCL19-expressing andCCL21-expressing cells; decreasing T cell proliferation induced by oneor more cells selected from dendritic cells, bone marrow-deriveddendritic cells, monocytes, microglia, M1 microglia, activated M1microglia, M2 microglia, macrophages, neutrophils, NK cells, M1macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages,activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2neutrophils, and M2 NK cells; inhibition of osteoclast production,decreased rate of osteoclastogenesis, or both; decreasing survival ofone or more cells selected from dendritic cells, bone marrow-deriveddendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; decreasing proliferation of one or morecells selected from dendritic cells, bone marrow-derived dendriticcells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; inhibiting migration of one or more cellsselected from dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibiting one or more functions of one or more cellsselected from dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibiting maturation of one or more cells selected fromdendritic cells, bone marrow-derived dendritic cells, macrophages,neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells,activated M1 macrophages, activated M1 neutrophils, activated M1 NKcells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibition of one or more types of clearance selected fromapoptotic neuron clearance, nerve tissue debris clearance, dysfunctionalsynapse clearance, non-nerve tissue debris clearance, bacteriaclearance, other foreign body clearance, disease-causing proteinclearance, disease-causing peptide clearance, and tumor cell clearance;optionally wherein the disease-causing protein is selected from amyloidbeta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursorprotein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUSprotein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein,prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase,ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10,Lewy body, atrial natriuretic factor, islet amyloid polypeptide,insulin, apolipoprotein AI, serum amyloid A, medin, prolactin,transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides and the tumor cell is from a cancer selected frombladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer; inhibition of phagocytosis of one or more of apoptoticneurons, nerve tissue debris, dysfunctional synapses, non-nerve tissuedebris, bacteria, other foreign bodies, disease-causing proteins,disease-causing peptides, disease-causing nucleic acids, or tumor cells;optionally wherein the disease-causing nucleic acids are antisenseGGCCCC (G2C4) repeat-expansion RNA, the disease-causing proteins areselected from amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides, and the tumor cells are from a cancer selectedfrom bladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, orthyroid cancer; binding to Siglec-9 ligand on tumor cells; binding toSiglec-9 ligand on cells selected from neutrophils, dendritic cells,bone marrow-derived dendritic cells, monocytes, microglia, macrophages,and NK cells; inhibition of tumor cell killing by one or more ofmicroglia, macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, neutrophils, T cells, T helper cells, orcytotoxic T cells; inhibiting anti-tumor cell proliferation activity ofone or more of microglia, macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, neutrophils, T cells, Thelper cells, or cytotoxic T cells; inhibition of anti-tumor cellmetastasis activity of one or more of microglia, macrophages,neutrophils, NK cells, dendritic cells, bone marrow-derived dendriticcells, neutrophils, T cells, T helper cells, or cytotoxic T cells;inhibition of one or more ITAM motif containing receptors, optionallywherein the one or more ITAM motif containing receptors are selectedfrom TREM1, TREM2, Sirp beta, FcgR, DAP10, and DAP12; inhibition ofsignaling by one or more pattern recognition receptors (PRRs),optionally wherein the one or more PRRs are selected from receptors thatidentify pathogen-associated molecular patterns (PAMPs), receptors thatidentify damage-associated molecular patterns (DAMPs), and anycombination thereof; inhibition of one or more receptors comprising themotif D/Ex₀₋₂YxxL/IX₆₋₈YxxL/I (SEQ ID NO: 252); inhibition of signalingby one or more Toll-like receptors; inhibition of the JAK-STAT signalingpathway; inhibition of nuclear factor kappa-light-chain-enhancer ofactivated B cells (NFκB); de-phosphorylation of an ITAM motif containingreceptor; modulated expression of one or more inflammatory receptors,proteins of the complement cascade, and/or receptors that are expressedon immune cells, optionally wherein the one or more inflammatoryreceptors, proteins of the complement cascade, and/or receptors that areexpressed on immune cells comprise CD86, C1qa, C1qB, C1qC, C1s, C1R, C4,C2, C3, ITGB2, HMOX1, LAT2, CASP1, CSTA, VSIG4, MS4A4A, C3AR1, GPX1,TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX, and/or PYCARD, and the oneor more inflammatory receptors, proteins of the complement cascade,and/or receptors that are expressed on immune cells are expressed on oneor more of microglia, macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, neutrophils, T cells, Thelper cells, or cytotoxic T cells; increasing expression of one or moreSiglec-9-dependent genes; normalization of disrupted Siglec-9-dependentgene expression; decreasing expression of one or more ITAM-dependentgenes, optionally wherein the one more ITAM-dependent genes areactivated by nuclear factor of activated T cells (NFAT) transcriptionfactors; promoting or rescuing functionality of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, and regulatory T cells;increasing infiltration of one or more of immunosuppressor dendriticcells, immunosuppressor macrophages, immunosuppressor neutrophils,immunosuppressor NK cells, myeloid-derived suppressor cells,tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, and regulatory T cells into tumors;increasing the number of tumor-promoting myeloid/granulocyticimmune-suppressive cells in a tumor, in peripheral blood, or otherlymphoid organ; enhancing tumor-promoting activity of myeloid-derivedsuppressor cells; increasing expression of tumor-promoting cytokines ina tumor or in peripheral blood, optionally wherein the tumor-promotingcytokines are TGF-beta or IL-10; increasing tumor infiltration oftumor-promoting FoxP3+ regulatory T lymphocytes; enhancingtumor-promoting activity of myeloid-derived suppressor cells (MDSC);decreasing activation of tumor-specific T lymphocytes with tumor killingpotential; decreasing infiltration of tumor-specific NK cells with tumorkilling potential; decreasing the tumor killing potential of NK cells;decreasing infiltration of tumor-specific B lymphocytes with potentialto enhance immune response; decreasing infiltration of tumor-specific Tlymphocytes with tumor killing potential; increasing tumor volume;increasing tumor growth rate; increasing metastasis; increasing rate oftumor recurrence; decreasing efficacy of one or more immune-therapiesthat modulate anti-tumor T cell responses, optionally wherein the one ormore immune-therapies are immune-therapies that target one or moretarget proteins selected from PD1/PDL1, CD40, OX40, ICOS, CD28,CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM,LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR,LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1 receptor, and anycombination thereof, or of one or more cancer vaccines; inhibition ofPLCγ/PKC/calcium mobilization; and inhibition of PI3K/Akt, Ras/MAPKsignaling.

In some embodiments, treatment of cancer with agents, such as Siglec-9blocking antibodies: (i) directly or indirectly decrease the survival,proliferation, maturation, differentiation, and/or functionality oftumor-promoting myeloid/granulocytic immune-suppressive cells thataccumulate in the tumor, in peripheral blood, and in lymphoid organs ofcancer patients; (ii) decrease the number of tumor-promotingmyeloid/granulocytic immune-suppressive cells in the tumor, in theperipheral blood, and in other lymphoid organs of a cancer patient;(iii) block tumor-promoting activity of myeloid-derived suppressor cells(MDSC); (iv) decrease expression of tumor-promoting cytokines, such asTGF-beta and IL-10, in the tumor and in the peripheral blood of a cancerpatient; (v) decrease tumor-promoting FoxP3+ regulatory T lymphocyteinfiltration in the tumor; (vi) increase infiltration and activation ofT lymphocytes with tumor killing potential; (vii) increase infiltrationof tumor-specific NK cells with tumor killing potential; (viii) increasethe tumor killing potential of NK cells; (ix) increase infiltration oftumor-specific B lymphocytes with potential to enhance immune response;(x) decrease tumor volume; (xi) reduce tumor growth rate; (xii) reduceand/or inhibit metastasis; (xiii) reduce rate of tumor recurrence; (xiv)increase efficacy of immune-therapy that modulates anti-tumor T cellresponses, such as PD1/PDL1, CTLA4, CD40, OX40, ICOS, CD28, CD137/4-1BB,CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA,KIR, GAL9, CD2, CD5, CD39, CD73, CD30, TIGIT, VISTA, TIM1, TIM3, TIM4,and cancer vaccines, (xv) induce, activate, or otherwise increasePLCγ/PKC/calcium mobilization; and (xvi) induce, activate, or otherwiseincrease PI3K/Akt, Ras/MAPK signaling.

Immunosuppressor cells are sometimes also referred to as myeloid-derivedsuppressor cells (MDSC). In humans, MDSCs can be defined by one of thefollowing combination of markers: (1) CD14⁺ HLA-DR^(low/−), (2) CD14⁺IL4Rα⁺, (3) CD14⁺ HLA-DR⁻ IL4Rα⁺, (4) CD34⁺CD14⁺CD11b⁺ Siglec-9⁺, (5)CD11b⁺ CD14⁺ Siglec-9⁺, (6) Siglec-9⁺ HLA-DR⁻, (7) Lin⁻ HLA-DR⁻, (8)Lin⁻ HLA-DR⁻ Siglec-9⁺, (9) Lin⁻ HLA-DR⁻ Siglec-9⁺CD11b⁺, (10) Lin⁻Siglec-9⁺CD11b⁺CD15⁺, (11) Lin⁻ HLA-DR⁻ Siglec-9⁺CD11b⁺CD14 CD15⁺, (12)CD11b⁺CD14 Siglec-9⁺, (13) CD11b⁺CD14⁻ HLA-DR⁻ Siglec-9⁺CD15⁺, (14)Siglec-9⁺ HLA-DR⁻ CD15⁺, (15) CD15⁺ IL4Rα⁺, (16) CD11b⁺CD15⁺CD66b⁺, (17)CD15⁺ FSC^(low) SSC^(high), (18) CD15high Siglec-9⁺, (19) CD11b⁺CD14⁻CD15⁺, (20) CD66b⁺ SSC^(high), and (21) CD11b⁺CD15⁺ (see also Solito Set al. Annals of the NY Academy of Sciences, 2014). In mice, MDSCs canbe defined by the expression of the surface markers CD45⁺, CD11b⁺, Gr1⁺,and/or Il4Rα⁺. Additional exemplary immunosuppressive monocytic lineagesare CD45⁺, CD11b⁺, Gr1^(low); and CD45⁺, CD11c⁺.

The present disclosure further relates to agents that bind or interactwith Siglec-9, such as anti-Siglec-9 antibodies. In certain embodiments,the anti-Siglec-9 antibodies do not significantly decrease cell surfacelevels of Siglec-9, and/or do not inhibit interaction between Siglec-9and one or more Siglec-9 ligands.

Siglec-9 Proteins

In one aspect, the present disclosure provides agents, such as isolated(e.g., monoclonal) antibodies, that interact with or otherwise bind toregions, such as epitopes, within a Siglec-9 protein of the presentdisclosure. In some embodiments, agents of the present disclosure, suchas anti-Siglec-9 antibodies of the present disclosure, bind to aSiglec-9 protein and modulate one or more Siglec-9 activities afterbinding to the Siglec-9 protein, for example, an activity associatedwith Siglec-9 expression in a cell. Siglec-9 proteins of the presentdisclosure include, without limitation, a mammalian Siglec-9 protein,human Siglec-9 protein, mouse Siglec-9 protein, and rat Siglec-9protein.

Siglec-9 is variously referred to as a Siglec-9 molecule, Sialicacid-binding Ig-like lectin 9, CD329 antigen, CD329; CDw329, FOAP-9, andOBBP-LIKE.

Siglec-9 is an immunoglobulin-like receptor primarily expressed onmyeloid lineage cells, including without limitation, macrophages,neutrophils, NK cells, dendritic cells, osteoclasts, monocytes, andmicroglia. In some embodiments, Siglec-9 forms a receptor-signalingcomplex with CD64. In some embodiments, Siglec-9 signaling results inthe downstream inhibition of PI3K or other intracellular signals. Onmyeloid cells, Toll-like receptor (TLR) signals are important for theinhibition of Siglec-9 activities, e.g., in the context of an infectionresponse. TLRs also play a key role in the pathological inflammatoryresponse, e.g., TLRs expressed in macrophages, neutrophils, NK cells anddendritic cells.

Various Siglec-9 homologs are known, including without limitation, humanSiglec-9, chimpanzee Siglec-9, green monkey Siglec-9, rhesus macaqueSiglec-9, and mouse Siglec-9. The amino acid sequence of human Siglec-9is set forth below as SEQ ID NO: 1:

        10         20         30         40         50MLLLLLPLLW GRERAEGQTS KLLTMQSSVT VQEGLCVHVP CSFSYPSHGW        60         70         80         90        100IYPGPVVHGY WFREGANTDQ DAPVATNNPA RAVWEETRDR FHLLGDPHTK       110        120        130        140        150NCTLSIRDAR RSDAGRYFFR MEKGSIKWNY KHHRLSVNVT ALTHRPNILI       160        170        180        190        200PGTLESGCPQ NLTCSVPWAC EQGTPPMISW IGTSVSPLDP STTRSSVLTL       210        220        230        240        250IPQPQDHGTS LTCQVTFPGA SVTTNKTVHL NVSYPPQNLT MTVFQGDGTV       260        270        280        290        300STVLGNGSSL SLPEGQSLRL VCAVDAVDSN PPARLSLSWR GLTLGPSQPS       310        320        330        340        350NPGVLELPWV HLRDAAEFTC RAQNPLGSQQ VYLNVSLQSK ATSGVTQGVV       360        370        380        390        400GGAGATALVF LSFCVIFVVV RSCRKKSARP AAGVGDTGIE DANAVRGSAS       410        420        430        440        450QGPLTEPWAE DSPPDQPPPA SARSSVGEGE LQYASLSFQM VKPWDSRGQE        460ATDTEYSEIK IHR

In some embodiments, the Siglec-9 is a preprotein that includes a signalsequence. In some embodiments, the Siglec-9 is a mature protein. In someembodiments, the mature Siglec-9 protein does not include a signalsequence. In some embodiments, the mature Siglec-9 protein is expressedon a cell. In some embodiments, the mature Siglec-9 protein is expressedon a cell, such as the surface of a cell, including, without limitation,human dendritic cells, human macrophages, human monocytes, humanosteoclasts, human neutrophils, human T cells, human helper T cell,human cytotoxic T cells, human granulocytes, and human microglia. Agentsof the present disclosure, such as anti-Siglec-9 antibodies of thepresent disclosure, may bind any of the Siglec-9 proteins of the presentdisclosure expressed on any cell disclosed herein.

Siglec-9 proteins of the present disclosure, such as human Siglec-9,contain several domains, including without limitation, a signal sequencelocated at amino acid residues 1-17 SEQ ID NO: 1, an extracellularimmunoglobulin-like variable-type (IgV) domain located at amino acidresidues 20-140 of SEQ ID NO: 1, two Ig-like C2-type domains located atamino acid residues 146-229 and 236-336 of SEQ ID NO: 1, a transmembranedomain located at amino acid residues 348-370 of SEQ ID NO: 1, an ITIMmotif located at amino acid residues 431-436 of SEQ ID NO: 1, andSLAM-like motif located at amino acid residues 454-459 of SEQ ID NO: 1.As one of skill in the art will appreciate, the beginning and endingresidues of the domains of the present disclosure may vary dependingupon the computer modeling program used or the method used fordetermining the domain.

Certain aspects of the present disclosure provide anti-Siglec-9antibodies that hind to a human Siglec-9, or a homolog thereof,including without limitation a mammalian Siglec-9 protein and Siglec-9orthologs from other species. Exemplary Siglec-9 homologs and orthologsare listed in Table A.

TABLE A Siglec-9 homologs and orthologs Organism Siglec-9 AccessionNumber Chimpanzee (Pan troglodytes) NCBI Accession No. XP_003316614Green monkey (Chlorocebus sabaeus) NCBI Accession No. XP_007995940.1Rhesus macaque (Macaca mulatta) NCBI Accession No. XP_001114560.2 Mouse(Mus musculus) NCBI Accession No. NP_112458.2

Accordingly, as used herein a “Siglec-9” protein of the presentdisclosure includes, without limitation, a mammalian Siglec-9 protein,human Siglec-9 protein, and primate Siglec-9 protein. Additionally,anti-Siglec-9 antibodies of the present disclosure may bind an epitopewithin one or more of a mammalian Siglec-9 protein, human Siglec-9protein, and primate Siglec-9. In some embodiments, anti-Siglec-9antibodies of the present disclosure may bind specifically to amammalian Siglec-9 protein, human Siglec-9 protein, or both. In certainembodiments, anti-Siglec-9 antibodies of the present disclosure may bindspecifically to human Siglec-9, primate Siglec-9, or both.

In some embodiments, agents of the present disclosure that decreasecellular levels of Siglec-9 and/or inhibit interaction between Siglec-9and one or more Siglec-9 ligands, or that bind or interact withSiglec-9, such as anti-Siglec-9 antibodies of the present disclosure,may bind Siglec-9 in a pH dependent manner. In some embodiments, agentsof the present disclosure, such as anti-Siglec-9 antibodies, can bind toSiglec-9 at a neutral pH and be internalized without dissociating fromthe Siglec-9 protein. Alternatively, at an acidic pH agents of thepresent disclosure, such as anti-Siglec-9 antibodies, may dissociatefrom Siglec-9 once they are internalized and are then degraded byendosome/lysosome pathway. In certain embodiments, an anti-Siglec-9antibody binds Siglec-9 at a pH that ranges from 5.5 to 8.0, from 5.5 to7.5, from 5.5 to 7.0, from 5.5 to 6.5, from 5.5 to 6.0, from 6.0 to 8.0,from 6.5 to 8.0, from 7.0 to 8.0, from 7.5 to 8.0, from 6.0 to 7.5, from6.0 to 7.0, from 6.5 to 7.5. In certain embodiments, an anti-Siglec-9antibody dissociates from Siglec-9 at a pH of less than 6.0, less than5.5, less than 5.0, less than 4.5, less than 4.0, less than 3.5, lessthan 3.0, less than 2.5, or less than 2.0.

In some embodiments, agents of the present disclosure that decreasecellular levels of Siglec-9 and/or inhibit interaction between Siglec-9and one or more Siglec-9 ligands, or that bind or interact withSiglec-9, such as anti-Siglec-9 antibodies of the present disclosure,bind to a wild-type Siglec-9 protein of the present disclosure,naturally occurring variants thereof, and/or disease variants thereof.

In some embodiments, agents of the present disclosure that decreasecellular levels of Siglec-9 and/or inhibit interaction between Siglec-9and one or more Siglec-9 ligands, or that bind or interact withSiglec-9, such as anti-Siglec-9 antibodies of the present disclosure,bind a variant of human Siglec-9.

In some embodiments, agents of the present disclosure that decreasecellular levels of Siglec-9 and/or inhibit interaction between Siglec-9and one or more Siglec-9 ligands, or that bind or interact withSiglec-9, such as anti-Siglec-9 antibodies of the present disclosure,bind to a Siglec-9 protein expressed on the surface of a cell including,without limitation, human dendritic cells, human macrophages, human NKcells, human monocytes, human osteoclasts, human neutrophils, human Tcells, human T helper cell, human cytotoxic T cells, human granulocytes,and human microglia. In some embodiments, agents of the presentdisclosure that decrease cellular levels of Siglec-9 and/or inhibitinteraction between Siglec-9 and one or more Siglec-9 ligands, or thatbind or interact with Siglec-9, such as anti-Siglec-9 antibodies of thepresent disclosure, bind to a Siglec-9 protein expressed on the surfaceof a cell and modulate (e.g., induce or inhibit) at least one Siglec-9activity of the present disclosure after binding to the surfaceexpressed Siglec-9 protein. In some embodiments of the presentdisclosure, the anti-Siglec-9 antibody binds specifically to a Siglec-9protein. In some embodiments of the present disclosure, theanti-Siglec-9 antibody further binds to at least one additional Siglecprotein. In some embodiments, the anti-Siglec-9 antibody modulates oneor more activities of the at least one additional Siglec protein or of acell expressing the at least one additional Siglec protein.

Siglec-9 Ligands

Siglec-9 proteins of the present disclosure can interact with (e.g.,bind to) one or more Siglec-9 ligands.

Exemplary Siglec-9 ligands include, without limitation, sialic acid,sialic acid-containing glycolipids, sialic acid-containingglycoproteins, alpha-2,8-disialyl containing glycolipids, branchedalpha-2,6-linked sialic acid-containing glycoproteins, terminalalpha-2,6-linked sialic acid-containing glycolipids, terminalalpha-2,3-linked sialic acid-containing glycoproteins,disialogangliosides (e.g., gangliosides or glycolipids containing aceramide linked to a sialylated glycan), secreted mucins, Siglec-9ligands expressed on red blood cells, Siglec-9 ligands expressed onbacterial cells, Siglec-9 ligands expressed on apoptotic cells, Siglec-9ligands expressed on nerve cells, Siglec-9 ligands expressed on gliacells, Siglec-9 ligands expressed on microglia, Siglec-9 ligandsexpressed on astrocytes, Siglec-9 ligands expressed on tumor cells,Siglec-9 ligands expressed on viruses, Siglec-9 ligands expressed ondendritic cells, Siglec-9 ligands bound to beta amyloid plaques,Siglec-9 ligands bound to Tau tangles, Siglec-9 ligands ondisease-causing proteins, Siglec-9 ligands on disease-causing peptides,Siglec-9 ligands expressed on macrophages, Siglec-9 ligands expressed onneutrophils, Siglec-9 ligands expressed on natural killer cells,Siglec-9 ligands expressed on monocytes, Siglec-9 ligands expressed on Tcells, Siglec-9 ligands expressed on T helper cells, Siglec-9 ligandsexpressed on cytotoxic T cells, Siglec-9 ligands expressed on B cells,Siglec-9 ligands expressed on tumor-imbedded immunosuppressor dendriticcells, Siglec-9 ligands expressed on tumor-imbedded immunosuppressormacrophages, Siglec-9 ligands expressed on myeloid-derived suppressorcells, Siglec-9 ligands expressed on regulatory T cells. In someembodiments, Siglec-9 ligands of the present disclosure are ganglioside(e.g., disialogangliosides). Disialogangliosides generally share acommon lacto-ceramide core and one or more sialic acid residues.

Further examples of suitable Siglec-9 ligands are depicted in FIG. 2.

Further examples of suitable ganglioside (e.g., disialogangliosides)ligands are depicted in FIG. 3 and listed in Table B. Generally, aganglioside (e.g., disialogangliosides) is a molecule composed of aglycosphingolipid with one or more sialic acids (e.g.,n-acetyl-neuraminic acid, NANA) linked on the sugar chain.

TABLE B Structures of exemplary ganglioside Siglec-9 ligands GM2-1 =aNeu5Ac(2-3)bDGalp(1-?)bDGalNAc(1-?)bDGalNAc(1-?)bDGlcp(1-1)Cer GM3 =aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer GM2, GM2a(?) =bDGalpNAc(1-4)[aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)Cer GM2b(?) =aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer GM1, GM1a =bDGalp(1-3)bDGalNAc[aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)Cer asialo-GM1,GA1 = bDGalp(1-3)bDGalpNAc(1-4)bDGalp(1-4)bDGlcp(1-1)Cer asialo-GM2, GA2= bDGalpNAc(1-4)bDGalp(1-4)bDGlcp(1-1)Cer GM1b =aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)bDGalp(1-4)bDGlcp(1-1)Cer GD3 =aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer GD2 =bDGalpNAc(1-4)[aNeu5Ac(2-8)aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)Cer GD1a =aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)CerGD1alpha =aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2-6)]bDGalp(1-4)bDGlcp(1-1)CerGD1b =bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2-8)aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)CerGT1a =aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)Cer GT1, GT1b =aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2-8)aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)Cer OAc-GT1b =aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)aXNeu5Ac9Ac(2-8)aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)Cer GT1c =bDGalp(1-3)bDGalNAc(1-4)[aNeu5Ac(2-8)aNeu5Ac(2-8)aNeu5Ac(2-3)]bDGalp(1-4)bDGlcp(1-1)Cer GT3 =aNeu5Ac(2-8)aNeu5Ac(2-8)aNeu5Ac(2-3)bDGal(1-4)bDGlc(1-1)CerGQ1b =aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-3)bDGalNAc(1-4)┌aNeu5Ac(2-8)aNeu5Ac(2-3)┐bDGalp(1-4)bDGlcp(1-1)CerGGal = aNeu5Ac(2-3)bDGalp(1-1)Cer where: aNeu5Ac =5-acetyl-alpha-neuraminic acid aNeu5Ac9Ac =5,9-diacetyl-alpha-neuraminic acid bDGalp = beta-D-galactopyranosebDGalpNAc = N-acetyl-beta-D-galactopyranose bDGlcp =beta-D-glucopyranose Cer = ceramide (general N-acylated sphingoid)Siglec-9 Agents

Certain aspects of the present disclosure relate to agents (e.g.,Siglec-9 agents) that decrease cellular levels of Siglec-9 and/orinhibit interaction between Siglec-9 and one or more Siglec-9 ligands.Other aspects of the present disclosure relate to agents (e.g., Siglec-9agents) that bind Siglec-9 without decreasing cellular levels ofSiglec-9 and/or without inhibiting interaction between Siglec-9 and oneor more Siglec-9 ligands. Further aspects of the present disclosurerelate to agents (e.g., Siglec-9 agents) that bind or interact withSiglec-9. In some embodiments, agents of the present disclosure block,inhibit, reduce, or interfere with one or more activities of a Siglec-9protein in vitro, in situ, and/or in vivo. In some embodiments, agentsof the present disclosure do not block, inhibit, reduce, or interferewith one or more activities of a Siglec-9 protein in vitro, in situ,and/or in vivo. In some embodiments, agents of the present disclosure,increase, activate or induce one or more activities of a Siglec-9protein in vitro, in situ, and/or in vivo.

In certain embodiments, agents of the present disclosure are agents(e.g., Siglec-9 agents) that decrease cellular levels of Siglec-9 and/orinhibit interaction between Siglec-9 and one or more Siglec-9 ligand. Anagent of the present disclosure that decreases cellular levels ofSiglec-9 and/or inhibits interaction between Siglec-9 and one or moreSiglec-9 ligands is a molecule having one or more of the followingcharacteristics: (1) inhibits or reduces one or more Siglec-9activities; (2) the ability to inhibit or reduce binding of a Siglec-9to one or more of its ligands; (3) the ability to reduce Siglec-9expression (such as at the mRNA level and/or at protein level) inSiglec-9-expressing cells; (4) the ability to interact, bind, orrecognize a Siglec-9 protein; (5) the ability to specifically interactwith or bind to a Siglec-9 protein; and (6) the ability to treat,ameliorate, or prevent any aspect of a disease or disorder described orcontemplated herein.

Exemplary agents that inhibit the production of Siglec-9 include,without limitation, compounds that specifically inhibit Siglec-9synthesis and/or release, antisense molecules directed to a Siglec-9, ora short interfering RNA (siRNA) molecule directed to a nucleic acidencoding a Siglec-9. Additional exemplary agents that inhibit one ormore Siglec-9 activities include, without limitation, anti-Siglec-9antibodies that specifically bind to a Siglec-9 protein, compounds thatspecifically inhibit one or more Siglec-9 activities such as smallmolecule inhibitors and/or peptide inhibitors, compounds thatspecifically inhibit Siglec-9 binding to one or more ligands, a Siglec-9structural analog, or an RNA or DNA aptamer that binds a Siglec-9. Insome embodiments, an agent that decreases cellular levels of Siglec-9and/or inhibits interaction between Siglec-9 and one or more Siglec-9ligands is an allosteric inhibitor. In some embodiments, an agent thatdecreases cellular levels of Siglec-9 and/or inhibits interactionbetween Siglec-9 and one or more Siglec-9 ligands is an orthostericinhibitor.

In certain embodiments, an agent that decreases cellular levels ofSiglec-9 and/or inhibits interaction between Siglec-9 and one or moreSiglec-9 ligands is a small molecule inhibitor, including, withoutlimitation, small peptides or peptide-like molecules, soluble peptides,and synthetic non-peptidyl organic or inorganic compounds. A smallmolecule inhibitor may have a molecular weight of any of about 100 toabout 20,000 daltons (Da), about 500 to about 15,000 Da, about 1000 toabout 10,000 Da. Methods for making and testing the inhibitory effect asmall molecule has on one or more Siglec-9 activities are well known inthe art and such methods can be used to assess the effect of the smallmolecule inhibitor on Siglec-9 activity. For example, any of the methodsand assays disclosed herein may be used to screen for small moleculeinhibitors that decrease cellular levels of Siglec-9 and/or inhibitinteraction between Siglec-9 and one or more Siglec-9 ligand.

In certain embodiments, an agent that decreases cellular levels ofSiglec-9 and/or inhibits interaction between Siglec-9 and one or moreSiglec-9 ligands is an anti-Siglec-9 antibody that hinds or physicallyinteracts with a Siglec-9. The antibody may have nanomolar or evenpicomolar affinities for the target antigen (e.g., Siglec-9). In certainembodiments, the Kd of the antibody is about 0.05 to about 100 nM. Forexample, Kd of the antibody is any of about 100 nM, about 50 nM, about10 nM, about 1 nM, about 900 pM, about 800 pM, about 790 pM, about 780pM, about 770 pM, about 760 pM, about 750 pM, about 740 pM, about 730pM, about 720 pM, about 710 pM, about 700 pM, about 650 pM, about 600pM, about 590 pM, about 580 pM, about 570 pM, about 560 pM, about 550pM, about 540 pM, about 530 pM, about 520 pM, about 510 pM, about 500pM, about 450 pM, about 400 pM, about 350 pM about 300 pM, about 290 pM,about 280 pM, about 270 pM, about 260 pM, about 250 pM, about 240 pM,about 230 pM, about 220 pM, about 210 pM, about 200 pM, about 150 pM,about 100 pM, or about 50 pM to any of about 2 pM, about 5 pM, about 10pM, about 15 pM, about 20 pM, or about 40 pM. Methods for thepreparation and selection of antibodies that interact and/or bind withspecificity to a Siglec-9 are described herein.

In certain embodiments, an agent that decreases cellular levels ofSiglec-9 and/or inhibits interaction between Siglec-9 and one or moreSiglec-9 ligands comprises at least one antisense molecule capable ofblocking or decreasing the expression of a functional Siglec-9 bytargeting nucleic acids encoding a Siglec-9. Nucleic acid sequences ofSiglec-9 are known in the art. For example, a human Siglec-9 can have anucleic acid sequence as shown in NCBI Accession number NM_001198558.1and a chimpanzee Siglec-9 can have a nucleic acid sequence as shown inNCBI Accession No. XM_003316566.3 and a mouse SIGLEC-E can have anucleic acid sequence as shown in NCBI Accession No. NM_031181.2.Methods are known for the preparation of antisense oligonucleotidemolecules and such methods can be used to prepare antisenseoligonucleotides that will specifically bind one or more of a Siglec-9mRNA without cross-reacting with other polynucleotides. Exemplary sitesof targeting include, but are not limited to, the initiation codon, the5′ regulatory regions, the coding sequence, including any conservedconsensus regions, and the 3′ untranslated region. In certainembodiments, the antisense oligonucleotides are about 10 to about 100nucleotides in length, about 15 to about 50 nucleotides in length, about18 to about 25 nucleotides in length, or more. In certain embodiments,the oligonucleotides further comprise chemical modifications to increasenuclease resistance and the like, such as, for example, phosphorothioatelinkages and 2′-O-sugar modifications known to those of ordinary skillin the art.

In certain embodiments, an agent that decreases cellular levels ofSiglec-9 and/or inhibits interaction between Siglec-9 and one or moreSiglec-9 ligands comprises at least one siRNA molecule capable ofblocking or decreasing the expression of a functional Siglec-9 bytargeting nucleic acids encoding a Siglec-9. Methods for preparation ofsiRNA molecules are well known in the art and such methods can be usedto prepare siRNA molecules that will specifically target a Siglec-9 mRNAwithout cross-reacting with other polynucleotides. siRNA molecules maybe generated by methods such as by typical solid phase oligonucleotidesynthesis, and often will incorporate chemical modifications to increasehalf-life and/or efficacy of the siRNA agent, and/or to allow for a morerobust delivery formulation. Alternatively, siRNA molecules aredelivered using a vector encoding an expression cassette forintracellular transcription of siRNA.

In certain embodiments, an agent that decreases cellular levels ofSiglec-9 and/or inhibits interaction between Siglec-9 and one or moreSiglec-9 ligands is an RNA or DNA aptamer that binds or physicallyinteracts with a Siglec-9, and blocks interactions between a Siglec-9and one or more of its ligands. In certain embodiments, the aptamercomprises at least one RNA or DNA aptamer that binds to a mature form ofSiglec-9.

In certain embodiments, an agent that decreases cellular levels ofSiglec-9 and/or inhibits interaction between Siglec-9 and one or moreSiglec-9 ligands comprises at least one Siglec-9 structural analog. Theterm Siglec-9 structural analog refers to compounds that have a similarthree dimensional structure as part of that of a Siglec-9 and which bindto one or more CD3 ligands under physiological conditions in vitro or invivo, wherein the binding at least partially inhibits a Siglec-9biological activity. Suitable Siglec-9 structural analogs can bedesigned and synthesized through molecular modeling of Siglec-9 bindingto a ligand, such as a Siglec-9 ligand of the present disclosure. TheSiglec-9 structural analogs can be monomers, dimers, or higher ordermultimers in any desired combination of the same or different structuresto obtain improved affinities and biological effects. In someembodiments, the agent binds to or interacts with an amino acid sequenceof a Siglec-9.

In certain embodiments, an agent that decreases cellular levels ofSiglec-9 and/or inhibits interaction between Siglec-9 and one or moreSiglec-9 ligands comprises a soluble Siglec-9 receptor protein, asoluble Siglec-9-Fc fusion protein (e.g., Siglec-9 immunoadhesin), asoluble Siglec receptor that binds to a Siglec-9 ligand, a Siglec-Fcfusion protein (e.g., Siglec immunoadhesin) that binds to a Siglec-9ligand. In certain embodiments, such agents bind one or more Siglec-9ligands and thereby prevent the interaction between a given Siglec-9ligand and a functional Siglec-9 receptor.

In certain embodiments, agents of the present disclosure are agents(e.g., Siglec-9 agents) that bind or interact with Siglec-9. Exemplaryagents that bind or interact with Siglec-9 include, without limitation,inert anti-Siglec-9 antibodies, agonist anti-Siglec-9 antibodies,Siglec-9 ligands, Siglec-9 ligand agonist fragments, Siglec-9immunoadhesins, Siglec-9 soluble receptors, Siglec-Fc fusion proteins(e.g., Siglec immunoadhesins), soluble Siglec receptors, Siglec-9 ligandmimetics, and small molecule compounds. A small molecule compound mayhave a molecular weight of any of about 100 to about 20,000 daltons(Da), about 500 to about 15,000 Da, about 1000 to about 10,000 Da.Methods for making and testing the effect an agent has on one or moreSiglec-9 activities are well known in the art and such methods can beused to assess the effect of the small molecule inhibitor on Siglec-9activity. For example, any of the methods and assays disclosed hereinmay be used to screen for small molecule inhibitors that bind orinteract with Siglec-9.

Assays

Agents that decrease cellular levels of Siglec-9 and/or inhibitinteraction between Siglec-9 and one or more Siglec-9 ligands may beidentified and/or characterized using methods well known in the art,such as, for example, radiolabeled inhibitor assays, optical assays,protein binding assays, biochemical screening assays, immunoassays, massshift measurement assays, fluorescence assays, and/or fluorogenicpeptide cleavage assays.

Binding Assays and Other Assays

In certain embodiments, agents that decrease cellular levels of Siglec-9and/or inhibit interaction between Siglec-9 and one or more Siglec-9ligands can be identified by techniques well known in the art fordetecting the presence of a Siglec-9 agent candidate's interactionand/or binding affinity to a Siglec-9.

In certain embodiments, agents that interact with a Siglec-9 can beidentified using a radiolabeled inhibitor assay. For example, a knownamount of a radiolabeled agent candidate may be incubated with a knownamount of immobilized Siglec-9 and a buffer. Subsequently, theimmobilized Siglec-9 may be washed with a buffer and the immobilizedSiglec-9 may be measured for the remaining presence of the radiolabeledSiglec-9 agent candidate using techniques known in the art, such as, forexample, a gamma counter. A measurement indicating the presence of aradiolabeled substance may indicate the radiolabeled agent candidate iscapable of interacting with and/or binding to Siglec-9.

In certain embodiments, an agent that interacts with a Siglec-9 may beidentified using an optical technique. An exemplary optical technique todetect a Siglec-9 agent may include, e.g., attaching Siglec-9 to acolorimetric resonant grafting surface, thereby shifting the wavelengthof reflected light due to changes in the optical path the light musttake, and subsequently measuring additional changes in the wavelength ofreflected light when a candidate agent is allowed to interact withSiglec-9. For example, no change in the measured wavelength of reflectedlight when an agent is incubated with Siglec-9 may indicate that theagent candidate is unable to interact with Siglec-9. Changes in themeasured wavelength of reflected light when an agent candidate isincubated with Siglec-9 may indicate that the agent candidate is capableof binding and/or interacting with Siglec-9.

In certain embodiments, an agent that interacts with a Siglec-9 may beidentified using a protein-binding assay. An exemplary protein-bindingassay to detect a Siglec-9 agent may include, e.g.,co-immunoprecipitation of a Siglec-9 in the presence of the agentcandidate. For example, a Siglec-9 may be incubated with the agentcandidate in buffer, and subsequently an immobilized molecule specificto capture a Siglec-9, such as, for example, an anti-Siglec-9 antibody,may be used to capture Siglec-9 in the presence of the agent candidateand bind the Siglec-9, potentially with an interacting agent candidate,during wash procedures known in the art. Subsequently, Siglec-9,potentially with an interacting agent candidate, can be released and thepresence of an agent candidate may be detected, based on the agentcandidate characteristics, by techniques, such as, for example, massspectrometry and/or Western blot.

In certain embodiments, an agent that interacts with a Siglec-9 may beidentified using a biochemical and/or an immunoassay assay well known inthe art. An exemplary technique may include, e.g., an assay toquantitatively measure changes in Siglec-9 concentration and/or proteinhalf-life using techniques, such as, for example, Western blot,immunostaining, and co-immunoprecipitation. For example, an agentcandidate may be incubated with a sample containing a Siglec-9, such asa cell expressing Siglec-9, and subsequently Siglec-9 protein quantityand/or cellular levels may be measured at points during a time coursestudy. Changes in protein quantity, cellular levels, and/or proteinhalf-life in comparison to a control treatment may indicate that theSiglec-9 agent candidate may be capable of altering Siglec-9 half-lifeand/or activity.

In certain embodiments, a mass shift measurement assay may be used toidentify an agent that interacts with a Siglec-9. An exemplary massshift measurement assay may include, e.g., detecting the presence of astrongly and/or covalently bound Siglec-9 agent by measuring a change inSiglec-9 mass when the agent candidate is interacting with Siglec-9 byusing instruments, such as, but not limited to, a mass spectrometer. Forexample, a mass shift assay may be performed on a whole protein and/or apeptide-based analysis, depending on the nature of the agent candidateinteraction. Detection of a mass shift correlating with the addition ofsaid agent candidate to Siglec-9 may indicate that the agent candidatemay be capable of interacting with or otherwise inhibiting a Siglec-9.Additionally, an exemplary mass shift measurement assay may include,e.g., detecting the addition of mass to Siglec-9 correlating with therespective agent candidate mass when the agent candidate is interactingwith Siglec-9 using techniques, such as, for example, surface plasmonresonance. For example, the change in the refractive index of light maybe measured and correlated with a change in mass of Siglec-9 attached toa sensor surface.

In certain embodiments, a chemical cross-linking assay may be used toidentify a Siglec-9 agent that interacts with a Siglec-9. For example,an agent candidate may be incubated with a Siglec-9, in vivo or invitro, with a molecule cross-linker capable of covalently linking anagent candidate interacting with Siglec-9 to said Siglec-9 molecule.Subsequently, techniques, such as, but not limited to, mass spectrometryand/or Western blot, may be used to identify an agent candidate that maybe capable of interacting with or otherwise inhibiting Siglec-9. Forexample, detection of Siglec-9covalently cross-linked with the agentcandidate may indicate that the agent candidate may be capable ofinteracting with or otherwise inhibiting Siglec-9.

In certain embodiments, agents that interact with a Siglec-9 may beidentified using a fluorescence assay. For example, a known amount of afluorescent agent candidate may be incubated with a known amount ofimmobilized Siglec-9 and a buffer. Subsequently, the immobilizedSiglec-9 may be washed with a buffer and the immobilized Siglec-9 may bemeasured for the remaining presence of a fluorescent Siglec-9 agentcandidate using techniques known in the art, such as, but not limitedto, fluorescence detection. A measurement indicating the presence of afluorescent substance may indicate the fluorescent agent candidate iscapable of interacting with and/or binding to Siglec-9.

Activity Assays

Assays known in the art and described herein (e.g., Examples 1-10) canbe used for identifying and testing biological activities of Siglec-9agents of the present disclosure. In some embodiments, assays fortesting the ability of Siglec-9 agents for modulating one or moreSiglec-9 activities are provided.

Anti-Siglec-9 Antibodies

Certain aspects of the present disclosure relate to anti-Siglec-9antibodies that decrease cellular levels of Siglec-9 and/or inhibitinteraction (e.g., binding) between Siglec-9 and one or more Siglec-9ligands. In some embodiments, the anti-Siglec-9 antibody decreasescellular levels of Siglec-9 without inhibiting the interaction (e.g.,binding) between Siglec-9 and one or more Siglec-9 ligands. In someembodiments, the anti-Siglec-9 antibody inhibits the interaction (e.g.,binding) between Siglec-9 and one or more Siglec-9 ligands. In someembodiments, the anti-Siglec-9 antibody decreases cellular levels ofSiglec-9 and inhibits the interaction (e.g., binding) between Siglec-9and one or more Siglec-9 ligands. Other aspects of the presentdisclosure relate to anti-Siglec-9 antibodies that bind Siglec-9 withoutdecreasing cellular levels of Siglec-9 and/or without inhibitinginteraction (e.g., binding) between Siglec-9 and one or more Siglec-9ligands.

As disclosed herein, Siglec-9 may be constitutively recycled on cells,and as such may recycle into the cell (e.g., endocytose) any agents(e.g., antibodies) that bind Siglec-9 on the cell surface. However, suchendocytosis may not lead to a decrease in cellular levels (e.g., cellsurface levels) of Siglec-9. While it has been shown that acute myeloidleukemia (AML) cells may mediate endocytosis of anti-Siglec-9 antibodiesbound to surface-expressed Siglec-9, no decrease in cellular levels ofSiglec-9 was demonstrated. Accordingly, certain aspects of the presentdisclosure relate to anti-Siglec-9 antibodies that not only bind to cellsurface-expressed Siglec-9, but also decrease cellular levels ofSiglec-9. In some embodiments, anti-Siglec-9 antibodies of the presentdisclosure bind cell surface-expressed Siglec-9 and are furtherendocytosed into the cell. In some embodiments, anti-Siglec-9 antibodiesof the present disclosure bind cell surface-expressed Siglec-9 withoutbeing endocytosed into the cell.

Cellular levels of Siglec-9 may refer to, without limitation, cellsurface levels of Siglec-9, intracellular levels of Siglec-9, and totallevels of Siglec-9. In some embodiments, a decrease in cellular levelsof Siglec-9 comprises decrease in cell surface levels of Siglec-9. Asused herein, an anti-Siglec-9 antibody decreases cell surface levels ofSiglec-9 if it induces a decrease of 21% or more in cell surface levelsof Siglec-9 as measured by any in vitro cell-based assays or suitable invivo model described herein or known in the art, for example utilizingflow cytometry, such as fluorescence-activated cell sorting (FACS), tomeasure cell surface levels of Siglec-9. In some embodiments, a decreasein cellular levels of Siglec-9 comprises a decrease in intracellularlevels of Siglec-9. As used herein, an anti-Siglec-9 antibody decreasesintracellular levels of Siglec-9 if it induces a decrease of 21% or morein intracellular levels of Siglec-9 as measured by any in vitrocell-based assays or suitable in vivo model described herein or known inthe art, for example immunostaining, Western blot analysis,co-immunoprecipitation, and cell cytometry. In some embodiments, adecrease in cellular levels of Siglec-9 comprises a decrease in totallevels of Siglec-9. As used herein, an anti-Siglec-9 antibody decreasestotal levels of Siglec-9 if it induces a decrease of 21% or more intotal levels of Siglec-9 as measured by any in vitro cell-based assaysor suitable in vivo model described herein or known in the art, forexample immunostaining, Western blot analysis, co-immunoprecipitation,and cell cytometry. In some embodiments, the anti-Siglec-9 antibodiesinduce Siglec-9 degradation, Siglec-9 cleavage, Siglec-9internalization, Siglec-9 shedding, and/or downregulation of Siglec-9expression. In some embodiments, cellular levels of Siglec-9 aremeasured on primary cells (e.g., dendritic cells, bone marrow-deriveddendritic cells, monocytes, microglia, and macrophages) or on cell linesutilizing an in vitro cell assay.

In some embodiments, anti-Siglec-9 antibodies of the present disclosuredecrease cellular levels of Siglec-9 by at least 21%, at least 22%, atleast 23%, at least 24%, at least 25%, at least 26%, at least 27%, atleast 28%, at least 29%, at least 30%, at least 31%, at least 32%, atleast 33%, at least 34%, at least 35%, at least 36%, at least 37%, atleast 38%, at least 39%, at least 40%, at least 41%, at least 42%, atleast 43%, at least 44%, at least 45%, at least 46%, at least 47%, atleast 48%, at least 49%, at least 50%, at least 51%, at least 52%, atleast 53%, at least 54%, at least 55%, at least 56%, at least 57%, atleast 58%, at least 59%, at least 60%, at least 61%, at least 62%, atleast 63%, at least 64%, at least 65%, at least 66%, at least 67%, atleast 68%, at least 69%, at least 70%, at least 71%, at least 72%, atleast 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more as compared to cellular levels ofSiglec-9 in the absence of the anti-Siglec-9 antibody.

Any in vitro cell-based assays or suitable in vivo model describedherein or known in the art may be used to measure inhibition ofinteraction (e.g., binding) between Siglec-9 and one or more Siglec-9ligands. In some embodiments, anti-Siglec-9 antibodies of the presentdisclosure inhibit interaction (e.g., binding) between Siglec-9 and oneor more Siglec-9 ligands by at least 21%, at least 22%, at least 23%, atleast 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 31%, at least 32%, at least 33%, atleast 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or more at saturating antibody concentrations (e.g., 67 nM)utilizing any in vitro assay or cell-based culture assay describedherein or known in the art.

In some embodiments, anti-Siglec-9 antibodies of the present disclosureinhibit cell surface clustering of Siglec-9. In some embodiments,anti-Siglec-9 antibodies of the present disclosure inhibit one or moreactivities of a Siglec-9 protein, including, without limitation,phosphorylation of Tyr-433 and Tyr-456 by a Src family tyrosine kinase,such as Syk, LCK, FYM, and/or ZAP70; recruitment of and binding to thetyrosine-specific protein phosphatases SHP1 and SHP2; recruitment of andbinding to PLC-gamma1, which acts as a guanine nucleotide exchangefactor for Dynamini-1; recruitment of and binding to SH2-domaincontaining protein (e.g., Crk1); recruitment of and binding to thespleen tyrosine kinase Syk; recruitment of and binding to SH3-SH2-SH3growth factor receptor-bound protein 2 (Grb2); recruitment of andbinding to multiple SH2-containing proteins; modulated expression of oneor more pro-inflammatory cytokines, optionally wherein the one or moreanti-inflammatory cytokines are selected from a group consisting FN-α4,IFN-beta, IL-1β, IL-1alpha, TNF-α, IL-6, IL-8, CRP, IL-20 familymembers, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-17, IL-18,IL-33, MCP-1, and MIP-1-beta; modulated expression of one or morepro-inflammatory cytokines in one or more cells selected frommacrophages, neutrophils, NK cells, dendritic cells, bone marrow-deriveddendritic cells, monocytes, osteoclasts, T cells, T helper cells,cytotoxic T cells, granulocytes, and microglial cells; modulatedexpression of one or more anti-inflammatory cytokines, optionallywherein the one or more anti-inflammatory cytokines are selected fromIL-4, IL-10, IL-13, IL-35, IL-16, TGF-beta, IL-1Rα, G-CSF, and solublereceptors for TNF, IFN-beta1a, IFN-beta1b, or IL-6; modulated expressionof one or more anti-inflammatory cytokines in one or more cells selectedfrom macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, monocytes, osteoclasts, T cells, Thelper cells, cytotoxic T cells, granulocytes, and microglial cells;modulate expression of one or more proteins selected from C1qa, C1qB,C1qC, C1s, C1R, C4, C2, C3, ITGB2, HMOX1, LAT2, CASP1, CSTA, VSIG4,MS4A4A, C3AR1, GPX1, TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX, andPYCARD; inhibition of extracellular signal-regulated kinase (ERK)phosphorylation; decreasing tyrosine phosphorylation on one or morecellular proteins, optionally, wherein the one or more cellular proteinscomprise ZAP-70 and the tyrosine phosphorylation occurs on Tyr-319 ofZAP-70; modulated expression of C—C chemokine receptor 7 (CCR7);inhibition of microglial cell chemotaxis toward CCL19-expressing andCCL21-expressing cells; decreasing T cell proliferation induced by oneor more cells selected from dendritic cells, bone marrow-deriveddendritic cells, monocytes, microglia, M1 microglia, activated M1microglia, M2 microglia, macrophages, neutrophils, NK cells, M1macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages,activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2neutrophils, and M2 NK cells; inhibition of osteoclast production,decreased rate of osteoclastogenesis, or both; decreasing survival ofone or more cells selected from dendritic cells, bone marrow-deriveddendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; decreasing proliferation of one or morecells selected from dendritic cells, bone marrow-derived dendriticcells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; inhibiting migration of one or more cellsselected from dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibiting one or more functions of one or more cellsselected from dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibiting maturation of one or more cells selected fromdendritic cells, bone marrow-derived dendritic cells, macrophages,neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells,activated M1 macrophages, activated M1 neutrophils, activated M1 NKcells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibition of one or more types of clearance selected fromapoptotic neuron clearance, nerve tissue debris clearance, dysfunctionalsynapse clearance, non-nerve tissue debris clearance, bacteriaclearance, other foreign body clearance, disease-causing proteinclearance, disease-causing peptide clearance, and tumor cell clearance;optionally wherein the disease-causing protein is selected from amyloidbeta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursorprotein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUSprotein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein,prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase,ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10,Lewy body, atrial natriuretic factor, islet amyloid polypeptide,insulin, apolipoprotein AI, serum amyloid A, medin, prolactin,transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides and the tumor cell is from a cancer selected frombladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer; inhibition of phagocytosis of one or more of apoptoticneurons, nerve tissue debris, dysfunctional synapses, non-nerve tissuedebris, bacteria, other foreign bodies, disease-causing proteins,disease-causing peptides, disease-causing nucleic acids, or tumor cells;optionally wherein the disease-causing nucleic acids are antisenseGGCCCC (G2C4) repeat-expansion RNA, the disease-causing proteins areselected from amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides, and the tumor cells are from a cancer selectedfrom bladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, orthyroid cancer; binding to Siglec-9 ligand on tumor cells; binding toSiglec-9 ligand on cells selected from neutrophils, dendritic cells,bone marrow-derived dendritic cells, monocytes, microglia, macrophages,and NK cells; inhibition of tumor cell killing by one or more ofmicroglia, macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, neutrophils, T cells, T helper cells, orcytotoxic T cells; inhibiting anti-tumor cell proliferation activity ofone or more of microglia, macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, neutrophils, T cells, Thelper cells, or cytotoxic T cells; inhibition of anti-tumor cellmetastasis activity of one or more of microglia, macrophages,neutrophils, NK cells, dendritic cells, bone marrow-derived dendriticcells, neutrophils, T cells, T helper cells, or cytotoxic T cells;modulated expression of one or more inflammatory receptors, optionallywherein the one or more inflammatory receptors comprise CD86 and the oneor more inflammatory receptors are expressed on one or more ofmicroglia, macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, neutrophils, T cells, T helper cells, orcytotoxic T cells; inhibition of one or more ITAM motif containingreceptors, optionally wherein the one or more ITAM motif containingreceptors are selected from TREM1, TREM2, Sirp beta, FcgR, DAP10, andDAP12; inhibition of signaling by one or more pattern recognitionreceptors (PRRs), optionally wherein the one or more PRRs are selectedfrom receptors that identify pathogen-associated molecular patterns(PAMPs), receptors that identify damage-associated molecular patterns(DAMPs), and any combination thereof; inhibition of one or morereceptors comprising the motif D/Ex₀₋₂YxxL/IX₆₋₈YxxL/I (SEQ ID NO: 252);inhibition of signaling by one or more Toll-like receptors; inhibitionof the JAK-STAT signaling pathway; inhibition of nuclear factorkappa-light-chain-enhancer of activated B cells (NFκB);de-phosphorylation of an ITAM motif containing receptor; modulatedexpression of one or more inflammatory receptors, proteins of thecomplement cascade, and/or receptors that are expressed on immune cells,optionally wherein the one or more inflammatory receptors, proteins ofthe complement cascade, and/or receptors that are expressed on immunecells comprise CD86, C1qa, C1qB, C1qC, C1s, C1R, C4, C2, C3, ITGB2,HMOX1, LAT2, CASP1, CSTA, VSIG4, MS4A4A, C3AR1, GPX1, TyroBP, ALOX5AP,ITGAM, SLC7A7, CD4, ITGAX, and/or PYCARD, and the one or moreinflammatory receptors, proteins of the complement cascade, and/orreceptors that are expressed on immune cells are expressed on one ormore of microglia, macrophages, neutrophils, NK cells, dendritic cells,bone marrow-derived dendritic cells, neutrophils, T cells, T helpercells, or cytotoxic T cells; increasing expression of one or moreSiglec-9-dependent genes; normalization of disrupted Siglec-9-dependentgene expression; decreasing expression of one or more ITAM-dependentgenes, optionally wherein the one more ITAM-dependent genes areactivated by nuclear factor of activated T cells (NFAT) transcriptionfactors; promoting or rescuing functionality of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, and regulatory T cells;increasing infiltration of one or more of immunosuppressor dendriticcells, immunosuppressor macrophages, immunosuppressor neutrophils,immunosuppressor NK cells, myeloid-derived suppressor cells,tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, and regulatory T cells into tumors;increasing the number of tumor-promoting myeloid/granulocyticimmune-suppressive cells in a tumor, in peripheral blood, or otherlymphoid organ; enhancing tumor-promoting activity of myeloid-derivedsuppressor cells; increasing expression of tumor-promoting cytokines ina tumor or in peripheral blood, optionally wherein the tumor-promotingcytokines are TGF-beta or IL-10; increasing tumor infiltration oftumor-promoting FoxP3+ regulatory T lymphocytes; enhancingtumor-promoting activity of myeloid-derived suppressor cells (MDSC);decreasing activation of tumor-specific T lymphocytes with tumor killingpotential; decreasing infiltration of tumor-specific NK cells with tumorkilling potential; decreasing the tumor killing potential of NK cells;decreasing infiltration of tumor-specific B lymphocytes with potentialto enhance immune response; decreasing infiltration of tumor-specific Tlymphocytes with tumor killing potential; increasing tumor volume;increasing tumor growth rate; increasing metastasis; increasing rate oftumor recurrence; decreasing efficacy of one or more immune-therapiesthat modulate anti-tumor T cell responses, optionally wherein the one ormore immune-therapies are immune-therapies that target one or moretarget proteins selected from PD1/PDL1, CD40, OX40, ICOS, CD28,CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM,LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR,LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1 receptor, and anycombination thereof, or of one or more cancer vaccines; inhibition ofPLCγ/PKC/calcium mobilization; inhibition of PI3K/Akt, Ras/MAPKsignaling; enhancement of infiltration of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages, myeloidderived suppressor cells, tumor-associated macrophages, immunosuppressorneutrophils, non-tumorigenic CD45⁺CD14⁺ myeloid cells, and regulatory Tcells into tumors; increase in the number of tumor-promotingmyeloid/granulocytic immune-suppressive cells in a tumor, in peripheralblood, or other lymphoid organ; (r) enhancing tumor-promoting activityof non-tumorigenic myeloid-derived suppressor cells and/ornon-tumorigenic CD45⁺CD14⁺ myeloid cells; enhancement of survival ofnon-tumorigenic myeloid-derived suppressor cells (MDSC) and/ornon-tumorigenic CD45⁺CD14⁺ myeloid cells; decrease in activation oftumor-specific T lymphocytes with tumor killing potential; (e)decreasing activation of CD45⁺CD3⁺ T lymphocytes with tumor killingpotential; decrease in infiltration of tumor-specific NK cells withtumor killing potential; decrease in infiltration of tumor-specific Blymphocytes with potential to enhance immune response; decrease ininfiltration of tumor-specific T lymphocytes with tumor killingpotential; and decrease in infiltration of CD45⁺CD3⁺ T lymphocytes.

In some embodiments, the anti-Siglec-9 antibodies inhibit interaction(e.g., binding) between a Siglec-9 protein of the present disclosure andone or more Siglec-9 ligands including, without limitation, Siglec-9ligands expressed on red blood cells, Siglec-9 ligands expressed onbacterial cells, Siglec-9 ligands expressed on apoptotic cells, Siglec-9ligands expressed on nerve cells, Siglec-9 ligands expressed on gliacells, Siglec-9 ligands expressed on microglia, Siglec-9 ligandsexpressed on astrocytes, Siglec-9 ligands expressed on tumor cells,Siglec-9 ligands expressed on viruses, Siglec-9 ligands expressed ondendritic cells, Siglec-9 ligands bound to beta amyloid plaques,Siglec-9 ligands bound to Tau tangles, Siglec-9 ligands ondisease-causing proteins, Siglec-9 ligands on disease-causing peptides,Siglec-9 ligands expressed on macrophages, Siglec-9 ligands expressed onneutrophils, Siglec-9 ligands expressed on natural killer cells,Siglec-9 ligands expressed on monocytes, Siglec-9 ligands expressed on Tcells, Siglec-9 ligands expressed on T helper cells, Siglec-9 ligandsexpressed on cytotoxic T cells, Siglec-9 ligands expressed on B cells,Siglec-9 ligands expressed on tumor-imbedded immunosuppressor dendriticcells, Siglec-9 ligands expressed on tumor-imbedded immunosuppressormacrophages, Siglec-9 ligands expressed on myeloid-derived suppressorcells, Siglec-9 ligands expressed on regulatory T cells, secretedmucins, sialic acid, sialic acid-containing glycolipids, sialicacid-containing glycoproteins, alpha-2,8-disialyl containingglycolipids, branched alpha-2,6-linked sialic acid-containingglycoproteins, terminal alpha-2,6-linked sialic acid-containingglycolipids, terminal alpha-2,3-linked sialic acid-containingglycoproteins, and gangliosides (e.g., disialogangliosides).

In some embodiments, anti-Siglec-9 antibodies of the present disclosurebind to a Siglec-9 protein of the present disclosure expressed on thesurface of cell and the naked antibodies inhibit interaction (e.g.,binding) between the Siglec-9 protein and one or more Siglec-9 ligands.In some embodiments, anti-Siglec-9 antibodies of the present disclosurethat bind to a Siglec-9 protein of the present disclosure inhibitinteraction (e.g., binding) between the Siglec-9 protein and one or moreSiglec-9 ligands by reducing the effective levels of Siglec-9 that isavailable to interact with these proteins either on the cell surface orinside the cell. In some embodiments, anti-Siglec-9 antibodies of thepresent disclosure that bind to a Siglec-9 protein of the presentdisclosure inhibit interaction (e.g., binding) between the Siglec-9protein and one or more Siglec-9 ligands by inducing degradation ofSiglec-9.

In some embodiments that may be combined with any of the precedingembodiments, the anti-Siglec-9 antibody exhibits one or more activitiesselected from the group consisting of consisting of: (a) increasing thenumber of tumor infiltrating CD3⁺ T cells; (b) decreasing cellularlevels of Siglec-9 in non-tumorigenic CD14+ myeloid cells, optionallywherein the non-tumorigenic CD14+ myeloid cells are tumor infiltratingcells or optionally wherein the non-tumorigenic CD14+ myeloid cells arepresent in blood; (c) reducing the number of non-tumorigenic CD14+myeloid cells, optionally wherein the non-tumorigenic CD14+ myeloidcells are tumor infiltrating cells or optionally wherein thenon-tumorigenic CD14+ myeloid cells are present in blood; (d) reducingPD-L1 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (e)reducing PD-L2 levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (f) reducing B7-H2 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (g) reducing B7-H3 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (h) reducing CD200R levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (i) reducingCD163 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (j)reducing CD206 levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (k) decreasing tumor growth rate of solid tumors; (l) reducingtumor volume; (m) increasing efficacy of one or more PD-1 inhibitors;(n) increasing efficacy of one or more checkpoint inhibitor therapiesand/or immune-modulating therapies, optionally wherein the one or morecheckpoint inhibitor therapies and/or immune-modulating therapies targetone or more of CTLA4, the adenosine pathway, PD-L1, PD-L2, PD-L1, PD-L2,OX40, TIM3, LAG3, or any combination thereof; (o) increasing efficacy ofone or more chemotherapy agents, optionally wherein the one or more ofthe chemotherapy agents are gemcitabine, capecitabine, anthracyclines,doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel(Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide(Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (p)increasing proliferation of T cells in the presence of non-tumorigenicmyeloid-derived suppressor cells (MDSC); (q) inhibiting differentiation,survival, and/or one or more functions of non-tumorigenicmyeloid-derived suppressor cells (MDSC); and (r) killingSiglec-9-expressing immunosuppressor non-tumorigenic myeloid cellsand/or non-tumorigenic CD14-expressing cells in solid tumors andassociated blood vessels when conjugated to a chemical or radioactivetoxin. In some embodiments that may be combined with any of thepreceding embodiments, the anti-Siglec-9 antibody is not conjugated toan agent, optionally wherein the agent is drug, toxin, chemotherapeutic,or radioisotope.

Other aspects of the present disclosure relate to anti-Siglec-9antibodies that do not significantly decrease cell surface levels ofSiglec-9 and/or do not inhibit interaction between Siglec-9 and one ormore Siglec-9 ligands.

As used herein, an anti-Siglec-9 antibody does not significantlydecrease cell surface levels of Siglec-9 if it decreases ligand bindingto Siglec-9 by less than 20% as compared to cellular levels of Siglec-9in the absence of the anti-Siglec-9 antibody utilizing any in vitrocell-based assays or suitable in vivo model described herein or known inthe art. In some embodiments, anti-Siglec-9 antibodies of the presentdisclosure decrease cell surface levels of Siglec-9 by less than 20%,less than 19%, less than 18%, less than 17%, less than 16%, less than15%, less than 14%, less than 13%, less than 12%, less than 11%, lessthan 10%, less than 9%, less than 8%, less than 7%, less than 6%, lessthan 5%, less than 4%, less than 3%, less than 2%, or less than 1% ascompared to cellular levels of Siglec-9 in the absence of theanti-Siglec-9 antibody.

As used herein, an anti-Siglec-9 antibody does not inhibit theinteraction (e.g., binding) between Siglec-9 and one or more Siglec-9ligands if it decreases ligand binding to Siglec-9 by less than 20% atsaturating antibody concentrations (e.g., 67 nM) utilizing any in vitroassay or cell-based culture assay described herein or known in the art.In some embodiments, anti-Siglec-9 antibodies of the present disclosureinhibit interaction (e.g., binding) between Siglec-9 and one or moreSiglec-9 ligands by less than 20%, less than 19%, less than 18%, lessthan 17%, less than 16%, less than 15%, less than 14%, less than 13%,less than 12%, less than 11%, less than 10%, less than 9%, less than 8%,less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,less than 2%, or less than 1% at saturating antibody concentrations(e.g., 67 nM) utilizing any in vitro assay or cell-based culture assaydescribed herein or known in the art.

As used herein, levels of Siglec-9 may refer to expression levels of thegene encoding Siglec-9; to expression levels of one or more transcriptsencoding Siglec-9; to expression levels of Siglec-9 protein; and/or tothe amount of Siglec-9 protein present within cells and/or on the cellsurface. Any methods known in the art for measuring levels of geneexpression, transcription, translation, and/or protein abundance orlocalization may be used to determine the levels of Siglec-9.

Additionally, anti-Siglec-9 antibodies of the present disclosure can beused to prevent, reduce risk of, or treat dementia, frontotemporaldementia, Alzheimer's disease, vascular dementia, mixed dementia,Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophiclateral sclerosis, Huntington's disease, taupathy disease, Nasu-Hakoladisease, stroke, acute trauma, chronic trauma, lupus, acute and chroniccolitis, rheumatoid arthritis, wound healing, Crohn's disease,inflammatory bowel disease, ulcerative colitis, obesity, malaria,essential tremor, central nervous system lupus, Behcet's disease,Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,Shy-Drager syndrome, progressive supranuclear palsy, cortical basalganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, and cancerincluding bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and/or Haemophilus influenza. In someembodiments, anti-Siglec-9 antibodies of the present disclosure can beused for inducing or promoting the survival, maturation, functionality,migration, or proliferation of one or more immune cells in an individualin need thereof; or for decreasing the activity, functionality, orsurvival of regulatory T cells, tumor-imbedded immunosuppressordendritic cells, tumor-imbedded immunosuppressor macrophages,myeloid-derived suppressor cells, tumor-associated macrophages, acutemyeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cell,and/or chronic myeloid leukemia (CML) cell in an individual in needthereof. In some embodiments, anti-Siglec-9 antibodies of the presentdisclosure are monoclonal antibodies.

In some embodiments, an isolated anti-Siglec-9 antibody of the presentdisclosure decreases cellular levels of Siglec-9 (e.g., cell surfacelevels, intracellular levels, and/or total levels). In some embodiments,an isolated anti-Siglec-9 antibody of the present disclosure inducesdownregulation of Siglec-9. In some embodiments, an isolatedanti-Siglec-9 antibody of the present disclosure induces cleavage ofSiglec-9. In some embodiments, an isolated anti-Siglec-9 antibody of thepresent disclosure induces internalization of Siglec-9. In someembodiments, an isolated anti-Siglec-9 antibody of the presentdisclosure induces shedding of Siglec-9. In some embodiments, anisolated anti-Siglec-9 antibody of the present disclosure inducesdegradation of Siglec-9. In some embodiments, an isolated anti-Siglec-9antibody of the present disclosure induces desensitization of Siglec-9.In some embodiments, an isolated anti-Siglec-9 antibody of the presentdisclosure acts as a ligand mimetic to transiently activate Siglec-9. Insome embodiments, an isolated anti-Siglec-9 antibody of the presentdisclosure acts as a ligand mimetic and transiently activates Siglec-9before inducing a decrease in cellular levels of Siglec-9 and/orinhibition of interaction (e.g., binding) between Siglec-9 and one ormore Siglec-9 ligands. In some embodiments, an isolated anti-Siglec-9antibody of the present disclosure acts as a ligand mimetic andtransiently activates Siglec-9 before inducing degradation of Siglec-9.In some embodiments, an isolated anti-Siglec-9 antibody of the presentdisclosure acts as a ligand mimetic and transiently activates Siglec-9before inducing cleavage of Siglec-9. In some embodiments, an isolatedanti-Siglec-9 antibody of the present disclosure acts as a ligandmimetic and transiently activates Siglec-9 before inducinginternalization of Siglec-9. In some embodiments, an isolatedanti-Siglec-9 antibody of the present disclosure acts as a ligandmimetic and transiently activates Siglec-9 before inducing shedding ofSiglec-9. In some embodiments, an isolated anti-Siglec-9 antibody of thepresent disclosure acts as a ligand mimetic and transiently activatesSiglec-9 before inducing downregulation of Siglec-9 expression. In someembodiments, an isolated anti-Siglec-9 antibody of the presentdisclosure acts as a ligand mimetic and transiently activates Siglec-9before inducing desensitization of Siglec-9.

In some embodiments, an isolated anti-Siglec-9 antibody of the presentdisclosure is a murine antibody. In some embodiments, an isolatedanti-Siglec-9 antibody of the present disclosure is a human antibody, ahumanized antibody, a bispecific antibody, a monoclonal antibody, amultivalent antibody, or a chimeric antibody. Exemplary descriptions ofsuch antibodies are found throughout the present disclosure.

In some embodiments, anti-Siglec-9 antibodies of the present disclosurebind to a human Siglec-9, or a homolog thereof, including withoutlimitation, a mammalian Siglec-9 protein, chimpanzee Siglec-9 protein(NCBI Accession No. XP_003316614), green money Siglec-9 protein (NCBIAccession No. XP_007995940.1), rhesus macaque Siglec-9 protein (NCBIAccession No. XP_001114560.2), and mouse SIGLEC-E protein (NCBIAccession No. NP_112458.2). In some embodiments, anti-Siglec-9antibodies of the present disclosure specifically bind to humanSiglec-9. In some embodiments, anti-Siglec-9 antibodies of the presentdisclosure specifically bind to primate Siglec-9. In some embodiments,anti-Siglec-9 antibodies of the present disclosure specifically bind toboth human Siglec-9 and primate Siglec-9.

In some embodiments, anti-Siglec-9 antibodies of the present disclosureare agonist antibodies or antagonist antibodies that bind to a Siglec-9protein of the present disclosure expressed on the surface of a cell andmodulate (e.g., induce or inhibit) one or more Siglec-9 activities ofthe present disclosure after binding to the surface-expressed Siglec-9protein. In some embodiments, anti-Siglec-9 antibodies of the presentdisclosure are inert antibodies.

In some embodiments, anti-Siglec-9 antibodies of the present disclosuredo not significantly reduce TREM2 expression, including, withoutlimitation, cell surface levels of TREM2, intracellular levels of TREM2,and/or total levels of TREM2. In some embodiments, an anti-Siglec-9antibody does not reduce cellular levels of TREM2 in vivo. In someembodiments, the cellular levels of TREM2 are measured on primary cellsselected from dendritic cells, bone marrow-derived dendritic cells,monocytes, microglia, macrophages, neutrophils, and NK cells, or on celllines, and wherein the cellular levels of TREM2 are measured utilizingan in vitro cell assay. As used herein, an anti-Siglec-9 antibody doesnot significantly reduce cTREM2 expression if it reduced TREM2 by lessthan 20% as compared to TREM2 expression in the absence of theanti-Siglec-9 antibody utilizing any in vitro cell-based assays orsuitable in vivo model described herein or known in the art. In someembodiments, anti-Siglec-9 antibodies of the present disclosure decreaseTREM2 expression by less than 20%, less than 19%, less than 18%, lessthan 17%, less than 16%, less than 15%, less than 14%, less than 13%,less than 12%, less than 11%, less than 10%, less than 9%, less than 8%,less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,less than 2%, or less than 1% as compared to TREM2 expression in theabsence of the anti-Siglec-9 antibody.

Anti-Siglec-9 Antibody-Binding Regions

Certain aspects of the preset disclosure provide anti-Siglec-9antibodies that bind to one or more amino acids within amino acidresidues 20-347, 20-140, 141-347, 146-347, 146-229, 236-336, or 146-347of human Siglec-9 (SEQ ID NO: 1), or within amino acid residues on aSiglec-9 homolog or ortholog corresponding to amino acid residues20-347, 20-140, 141-347, 146-347, 146-229, 236-336, or 146-347 of SEQ IDNO: 1. In some embodiments, the anti-Siglec-9 antibody binds to one ormore amino acids within amino acid residues 62-76 of human Siglec-9 (SEQID NO: 1), or within amino acid residues on a Siglec-9 homolog orortholog corresponding to amino acid residues 62-76 of SEQ ID NO: 1. Insome embodiments, the anti-Siglec-9 antibody binds to one or more aminoacids within amino acid residues 62-76 and 86-92 of human Siglec-9 (SEQID NO: 1), or within amino acid residues on a Siglec-9 homolog orortholog corresponding to amino acid residues 62-76 and 86-92 of SEQ IDNO: 1. In some embodiments, the anti-Siglec-9 antibody hinds to one ormore amino acids within amino acid residues 86-92 of human Siglec-9 (SEQID NO: 1), or within amino acid residues on a Siglec-9 homolog orortholog corresponding to amino acid residues 86-92 of SEQ ID NO: 1. Insome embodiments, the anti-Siglec-9 antibody binds to one or more aminoacids within amino acid residues 86-96 of human Siglec-9 (SEQ ID NO: 1),or within amino acid residues on a Siglec-9 homolog or orthologcorresponding to amino acid residues 86-96 of SEQ ID NO: 1. In someembodiments, the anti-Siglec-9 antibody binds to one or more amino acidswithin amino acid residues 86-96 and 105-116 of human Siglec-9 (SEQ IDNO: 1), or within amino acid residues on a Siglec-9 homolog or orthologcorresponding to amino acid residues 86-96 and 105-116 of SEQ ID NO: 1.In some embodiments, the anti-Siglec-9 antibody binds to one or moreamino acids within amino acid residues 105-116 of human Siglec-9 (SEQ IDNO: 1), or within amino acid residues on a Siglec-9 homolog or orthologcorresponding to amino acid residues 105-116 of SEQ ID NO: 1. In someembodiments, the anti-Siglec-9 antibody binds to one or more amino acidswithin amino acid residues 107-115 of human Siglec-9 (SEQ ID NO: 1), orwithin amino acid residues on a Siglec-9 homolog or orthologcorresponding to amino acid residues 107-115 of SEQ ID NO: 1. In someembodiments, the anti-Siglec-9 antibody binds to one or more amino acidswithin amino acid residues 185-194 of human Siglec-9 (SEQ ID NO: 1), orwithin amino acid residues on a Siglec-9 homolog or orthologcorresponding to amino acid residues 185-194 of SEQ ID NO: 1.

In some embodiments, an anti-Siglec-9 antibody of the present disclosurebinds to one or more amino acid residues selected from L22, H48, W50,I51, Y52, K123, I126, D189, P190, R194 of SEQ ID NO: 1, or one or moreamino acid residues on a mammalian Siglec-9 protein corresponding to anamino acid residue selected from L22, H48, W50, I51, Y52, K123, I126,D189, P190, R194 of SEQ ID NO: 1. In some embodiments, an anti-Siglec-9antibody of the present disclosure binds to one or more, two or more, orall three amino acid residues selected from D189, P190, and R194 of SEQID NO: 1, or one or more, two or more, or all three amino acid residueson a mammalian Siglec-9 protein corresponding to an amino acid residueselected from D189, P190, and R194 of SEQ ID NO: 1. In some embodiments,an anti-Siglec-9 antibody of the present disclosure binds to one ormore, two or more, three or more, four or more, or all five amino acidresidues selected from H48, W50, I51, Y52, and 1126 of SEQ ID NO: 1, orone or more, two or more, three or more, four or more, or all five aminoacid residues on a mammalian Siglec-9 protein corresponding to an aminoacid residue selected from H48, W50, I51, Y52, and 1126 of SEQ ID NO: 1.In some embodiments, an anti-Siglec-9 antibody of the present disclosurebinds to one or more, two or more, three or more, four or more, five ormore, or all six amino acid residues selected from L22, H48, W50, I51,Y52, and K123 of SEQ ID NO: 1, or one or more, two or more, three ormore, four or more, five or more, or all six amino acid residues on amammalian Siglec-9 protein corresponding to an amino acid residueselected from L22, H48, W50, I51, Y52, and K123 of SEQ ID NO: 1.

As indicated in Table 8B, the critical Siglec-9 residues involved inbinding by antibody 2D4 corresponded to amino acid residues D189, P190,and R194 of SEQ ID NO: 1. The critical Siglec-9 residues involved inbinding by antibody 12B 12 corresponded to amino acid residues H48, W50,I51, Y52, and 1126 of SEQ ID NO: 1. The critical Siglec-9 residuesinvolved in binding by antibody 5C6 corresponded to amino acid residuesL22, H48, W50, I51, Y52, and K123 of SEQ ID NO: 1.

Other aspects of the preset disclosure provide anti-Siglec-9 antibodiesthat decrease cellular levels of Siglec-9 and/or inhibit interaction(e.g., binding) between Siglec-9, and that bind one or more Siglec-9ligands bind to one or more amino acids within amino acid residues62-76, 62-76 and 86-92, 86-92, 86-96, 86-96 and 105-116, 105-116, or107-115 of human Siglec-9 (SEQ ID NO: 1), or within amino acid residueson a Siglec-9 homolog or ortholog corresponding to amino acid residues62-76, 62-76 and 86-92, 86-92, 86-96, 86-96 and 105-116, 105-116, or107-115 of SEQ ID NO: 1.

Other aspects of the preset disclosure provide anti-Siglec-9 antibodiesthat do not significantly decrease cell surface levels of Siglec-9and/or do not inhibit interaction (e.g., binding) between Siglec-9 andone or more Siglec-9 ligands, and that bind to one or more amino acidswithin amino acid residues 185-194 of human Siglec-9 (SEQ ID NO: 1), orwithin amino acid residues on a Siglec-9 homolog or orthologcorresponding to amino acid residues 185-194 of SEQ ID NO: 1. Otheraspects of the preset disclosure provide anti-Siglec-9 antibodies thatdo not significantly decrease cell surface levels of Siglec-9 and/or donot inhibit interaction (e.g., binding) between Siglec-9 and one or moreSiglec-9 ligands, and that hind to one or more amino acids within aminoacid residues 62-76 of human Siglec-9 (SEQ ID NO: 1), or within aminoacid residues on a Siglec-9 homolog or ortholog corresponding to aminoacid residues 6 of SEQ ID NO: 1.

In some embodiments, anti-Siglec-9 antibodies of the present disclosuremay bind a conformational epitope. In some embodiments, anti-Siglec-9antibodies of the present disclosure may bind a discontinuous Siglec-9epitope. In some embodiments, the discontinuous Siglec-9 epitope mayhave two or more peptides, three or more peptides, four or morepeptides, five or more peptides, six or more peptides, seven or morepeptide, eight or more peptides, nine or more peptides, or 10 or morepeptides. As disclosed herein, Siglec-9 epitopes may comprise one ormore peptides comprising five or more, six or more, seven or more, eightor more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more 14or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or20 or more amino acid residues of the amino acid sequence of SEQ ID NO:1, or five or more, six or more, seven or more, eight or more, nine ormore, 10 or more, 11 or more, 12 or more, 13 or more 14 or more, 15 ormore, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or moreamino acid residues on a mammalian Siglec-9 protein corresponding to theamino acid sequence of SEQ ID NO: 1.

In some embodiments, anti-Siglec-9 antibodies of the present disclosurecompetitively inhibit binding of at least one antibody selected from anyof the antibodies listed in Tables 2, 3, 4A, 4B, 7A, and 7B. In someembodiments, anti-Siglec-9 antibodies of the present disclosurecompetitively inhibit binding of at least one antibody selected from2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2.

In some embodiments, anti-Siglec-9 antibodies of the present disclosurebind to an epitope of human Siglec-9 that is the same as or overlapswith the Siglec-9 epitope bound by at least one antibody selected fromany of the antibodies listed in Tables 2, 3, 4A, 4B, 7A, and 7B. In someembodiments, anti-Siglec-9 antibodies of the present disclosure bind toan epitope of human Siglec-9 that is the same as or overlaps with theSiglec-9 epitope bound by at least one antibody selected from 2D4, 2D5,5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2.

In some embodiments, anti-Siglec-9 antibodies of the present disclosurebind essentially the same Siglec-9 epitope bound by at least oneantibody selected from any of the antibodies listed in Tables 2, 3, 4A,4B, 7A, and 7B. In some embodiments, anti-Siglec-9 antibodies of thepresent disclosure bind essentially the same Siglec-9 epitope bound byat least one antibody selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6,12B12, and 17C2. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.).

In some embodiments, anti-Siglec-9 antibodies of the present disclosurecompete with one or more antibodies selected from 2D4, 2D5, 5B1, 6B2,6D8, 7H12, 5C6, 12B12, 17C2, and any combination thereof for binding toSiglec-9 when the anti-Siglec-9 antibody reduces the binding of one ormore antibodies selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12,17C2, and any combination thereof to Siglec-9 by an amount the rangesfrom about 50% to 100%, as compared to binding to Siglec-9 in theabsence of the anti-Siglec-9 antibody. In some embodiments, ananti-Siglec-9 antibody of the present disclosure competes with one ormore antibodies selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12,17C2, and any combination thereof for binding to Siglec-9 when theanti-Siglec-9 antibody reduces the binding of one or more antibodiesselected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, 17C2, and anycombination thereof to Siglec-9 by at least 50%, at least 55%, by atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100%, as compared to bindingto Siglec-9 in the absence of the anti-Siglec-9 antibody. In someembodiments, an anti-Siglec-9 antibody of the present disclosure thatreduces the binding of one or more antibodies selected from 2D4, 2D5,5B1, 6B2, 6D8, 7H12, 5C6, 12B12, 17C2, and any combination thereof toSiglec-9 by 100% indicates that the anti-Siglec-9 antibody essentialcompletely blocks the binding of one or more antibodies selected from2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, 17C2, and any combinationthereof to Siglec-9. In some embodiments, the anti-Siglec-9 antibody andthe one or more antibodies selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12,5C6, 12B12, 17C2, and any combination thereof are present in an amountthat corresponds to a 10:1 ratio, 9:1 ratio, 8:1 ratio, 7:1 ratio, 6:1ratio, 5:1 ratio, 4:1 ratio, 3:1 ratio, 2:1 ratio, 1:1 ratio, 0.75:1ratio, 0.5:1 ratio, 0.25:1 ratio, 0.1:1 ratio, 0.075:1 ratio, 0.050:1ratio, 0.025:1 ratio, 0.01:1 ratio, 0.0075: ratio, 0.0050:1 ratio,0.0025:1 ratio, 0.001: ratio, 0.00075:1 ratio, 0.00050:1 ratio,0.00025:1 ratio, 0.0001: ratio, 1:10 ratio, 1:9 ratio, 1:8 ratio, 1:7ratio, 1:6 ratio, 1:5 ratio, 1:4 ratio, 1:3 ratio, 1:2 ratio, 1:0.75ratio, 1:0.5 ratio, 1:0.25 ratio, 1:0.1 ratio, 1:0.075 ratio, 1:0.050ratio, 1:0.025 ratio, 1:0.01 ratio, 1:0.0075 ratio, 1:0.0050 ratio,1:0.0025 ratio, 1:0.001 ratio, 1:0.00075 ratio, 1:0.00050 ratio,1:0.00025 ratio, or 1:0.0001 ratio of anti-Siglec-9 antibody to one ormore antibodies selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12,17C2, and any combination thereof. In some embodiments, theanti-Siglec-9 antibody is present in excess by an amount that rangesfrom about 1.5-fold to 100-fold, or greater than 100-fold compared tothe amount of the one or more antibodies selected from 2D4, 2D5, 5B1,6B2, 6D8, 7H12, 5C6, 12B12, 17C2, and any combination thereof. In someembodiments, the anti-Siglec-9 antibody is present in an amount that isabout a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold,50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold,90-fold, 95-fold, or 100-fold excess compared to the amount of the oneor more antibodies selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6,12B12, 17C2, and any combination thereof.

Any suitable competition assay or Siglec-9 binding assay known in theart, such as BIAcore analysis, ELISA assays, or flow cytometry, may beutilized to determine whether an anti-Siglec-9 antibody competes withone or more antibodies selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6,12B 12, 17C2, and any combination thereof for binding to Siglec-9. In anexemplary competition assay, immobilized Siglec-9 or cells expressingSiglec-9 on the cell surface are incubated in a solution comprising afirst labeled antibody that binds to Siglec-9 (e.g., human or non-humanprimate) and a second unlabeled antibody that is being tested for itsability to compete with the first antibody for binding to Siglec-9. Thesecond antibody may be present in a hybridoma supernatant. As a control,immobilized Siglec-9 or cells expressing Siglec-9 is incubated in asolution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to Siglec-9, excess unbound antibody isremoved, and the amount of label associated with immobilized Siglec-9 orcells expressing Siglec-9 is measured. If the amount of label associatedwith immobilized Siglec-9 or cells expressing Siglec-9 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to Siglec-9. See, Harlow and Lane (1988) Antibodies: ALaboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

Anti-Siglec-9 Antibody Light Chain and Heavy Chain Variable Regions

In some embodiments, anti-Siglec-9 antibodies of the present disclosurecomprise (a) a light chain variable region comprising at least one, two,or three HVRs selected from HVR-L1, HVR-L2, and HVR-L3 of any one of theantibodies listed in Tables 2, 3, 4A, 4B, 7A, and 7B, or selected from2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, 17C2, and any combinationthereof; and/or (b) a heavy chain variable region comprising at leastone, two, or three HVRs selected from HVR-H1, HVR-H2, and HVR-H3 of anyone of the antibodies listed in Tables 2, 3, 4A, 4B, 7A, and 7B, orselected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, 17C2, and anycombination thereof. In some embodiments, the HVR-L1, HVR-L2, HVR-L3,HVR-H1, HVR-H2, and HVR-H3 comprise EU or Kabat CDR, Chothia CDR, orContact CDR sequences as shown in Tables 2, 3, 4A, 4B, 7A, and 7B, orfrom an antibody selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6,12B12, 17C2, and any combination thereof.

In some embodiments, anti-Siglec-9 antibodies of the present disclosurecomprise at least one, two, three, four, five, or six HVRs selected from(i) HVR-L1 comprising the amino acid sequence of any of the HVR-L1sequences listed in Tables 2, 3, 4A, 4B, 7A, and 7B, or from an antibodyselected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2; (ii)HVR-L2 comprising the amino acid sequence of any of the HVR-L2 sequenceslisted in Tables 2, 3, 4A, 4B, 7A, and 7B, or from an antibody selectedfrom 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2; (iii) HVR-L3comprising the amino acid sequence of any of the HVR-L3 sequences listedin Tables 2, 3, 4A, 4B, 7A, and 7B, or from an antibody selected from2D4, 2D5, 5B 1, 6B2, 6D8, 7H12, 5C6, 12B 12, and 17C2; (iv) HVR-H1comprising the amino acid sequence of any of the HVR-H1 sequences listedin Tables 2, 3, 4A, 4B, 7A, and 7B, or from an antibody selected from2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2; (v) HVR-H2comprising the amino acid sequence of any of the HVR-H2 sequences listedin Tables 2, 3, 4A, 4B, 7A, and 7B, or from an antibody selected from2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2; and (vi) HVR-H3comprising the amino acid sequence of any of the HVR-H3 sequences listedin Tables 2, 3, 4A, 4B, 7A, and 7B, or from an antibody selected from2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2.

In some embodiments, anti-Siglec-9 antibodies of the present disclosurecomprise a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain comprises one or moreof: (a) an HVR-L1 comprising an amino acid sequence selected from SEQ IDNOs: 6-9, 172, and 173, or an amino acid sequence with at least about90% homology to an amino acid sequence selected from SEQ ID NOs: 6-9,172, and 173; (b) an HVR-L2 comprising an amino acid sequence selectedfrom SEQ ID NOs: 10-13, 174, and 175, or an amino acid sequence with atleast about 90% homology to an amino acid sequence selected from SEQ IDNOs: 10-13, 174, and 175; and (c) an HVR-L3 comprising an amino acidsequence selected from SEQ ID NOs: 14-18, 176, and 177, or an amino acidsequence with at least about 90% homology to an amino acid sequenceselected from SEQ ID NOs: 14-18, 176, and 177; and/or wherein the heavychain variable domain comprises one or more of: (a) an HVR-H1 comprisingan amino acid sequence selected from SEQ ID NOs: 19-21, 178, and 179, oran amino acid sequence with at least about 90% homology to an amino acidsequence selected from SEQ ID NOs: 19-21, 178, and 179; (b) an HVR-H2comprising an amino acid sequence selected from SEQ ID NOs: 22-25, 180,and 181, or an amino acid sequence with at least about 90% homology toan amino acid sequence selected from SEQ ID NOs: 22-25, 180, and 181;and (c) an HVR-H3 comprising an amino acid sequence selected from SEQ IDNOs: 26-29, 182, and 183, or an amino acid sequence with at least about90% homology to an amino acid sequence selected from SEQ ID NOs: 26-29,182, and 183.

In some embodiments, anti-Siglec-9 antibodies of the present disclosurecomprise a light chain variable domain and a heavy chain variabledomain, wherein (a) the HVR-L1 comprises the amino acid sequence of SEQID NO: 6, the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 10,the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 14, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 19, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 22, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 26; (b) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 7, the HVR-L2 comprisesthe amino acid sequence of SEQ ID NO: 11, the HVR-L3 comprises the aminoacid sequence of SEQ ID NO: 15, the HVR-H1 comprises the amino acidsequence of SEQ ID NO: 20, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO: 23, and the HVR-H3 comprises the amino acid sequence ofSEQ ID NO: 27; (c) the HVR-L1 comprises the amino acid sequence of SEQID NO: 8, the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 12,the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 16, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 21, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 24, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 28; (d) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 9, the HVR-L2 comprisesthe amino acid sequence of SEQ ID NO: 13, the HVR-L3 comprises the aminoacid sequence of SEQ ID NO: 17, the HVR-H1 comprises the amino acidsequence of SEQ ID NO: 21, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO: 25, and the HVR-H3 comprises the amino acid sequence ofSEQ ID NO: 29; (e) the HVR-L1 comprises the amino acid sequence of SEQID NO: 8, the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 12,the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18, theHVR-H1 comprises the amino acid sequence of SEQ ID NO: 21, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 24, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 28; (f) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 172, the HVR-L2comprises the amino acid sequence of SEQ ID NO: 174, the HVR-L3comprises the amino acid sequence of SEQ ID NO: 176, the HVR-H1comprises the amino acid sequence of SEQ ID NO: 178, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 180, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 182; or (g) the HVR-L1comprises the amino acid sequence of SEQ ID NO: 173, the HVR-L2comprises the amino acid sequence of SEQ ID NO: 175, the HVR-L3comprises the amino acid sequence of SEQ ID NO: 177, the HVR-H1comprises the amino acid sequence of SEQ ID NO: 179, the HVR-H2comprises the amino acid sequence of SEQ ID NO: 181, and the HVR-H3comprises the amino acid sequence of SEQ ID NO: 183.

In some embodiments, anti-Siglec-9 antibodies of the present disclosurecomprise a light chain variable region of any one of the antibodieslisted in Tables 2, 3, 4A, 4B, 7A, and 7B, or selected from 2D4, 2D5,5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2; and/or a heavy chain variableregion of any one of the antibodies listed in Tables 2, 3, 4A, 4B, 7A,and 7B, or selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and17C2. In some embodiments, anti-Siglec-9 antibodies of the presentdisclosure comprise a light chain variable region comprising an aminoacid sequence selected from any of SEQ ID NOs: 61-115 and 197-204;and/or a heavy chain variable domain comprising an amino acid sequenceselected from any of SEQ ID NOs: 116-170 and 205-212. In someembodiments, the light chain variable domain comprises the amino acidsequence of SEQ ID NO: 61; and the heavy chain variable domain comprisesthe amino acid sequence of SEQ ID NO: 116. In some embodiments, thelight chain variable domain comprises the amino acid sequence of SEQ IDNO: 72; and the heavy chain variable domain comprises the amino acidsequence of SEQ ID NO: 127. In some embodiments, the light chainvariable domain comprises the amino acid sequence of SEQ ID NO: 83; andthe heavy chain variable domain comprises the amino acid sequence of SEQID NO: 138. In some embodiments, the light chain variable domaincomprises the amino acid sequence of SEQ ID NO: 94; and the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO: 149. Insome embodiments, the light chain variable domain comprises the aminoacid sequence of SEQ ID NO: 105; and the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO: 160. In someembodiments, the light chain variable domain comprises the amino acidsequence of SEQ ID NO: 197; and the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO: 205. In someembodiments, the light chain variable domain comprises the amino acidsequence of SEQ ID NO: 201; and the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO: 210.

In some embodiments, the anti-Siglec-9 antibody comprises the heavychain variable domain of antibody 5C6-H1 and the light chain variabledomain of antibody 5C6-L1. In some embodiments, the anti-Siglec-9antibody comprises the heavy chain variable domain of antibody 5C6-H2and the light chain variable domain of antibody 5C6-L2. In someembodiments, the anti-Siglec-9 antibody comprises the heavy chainvariable domain of antibody 5C6-H2 and the light chain variable domainof antibody 5C6-L3. In some embodiments, the anti-Siglec-9 antibodycomprises the heavy chain variable domain of antibody 5C6-H3 and thelight chain variable domain of antibody 5C6-L2. In some embodiments, theanti-Siglec-9 antibody comprises the heavy chain variable domain ofantibody 5C6-H3 and the light chain variable domain of antibody 5C6-L3.In some embodiments, the anti-Siglec-9 antibody comprises the heavychain variable domain of antibody 5C6-H4 and the light chain variabledomain of antibody 5C6-L2. In some embodiments, the anti-Siglec-9antibody comprises the heavy chain variable domain of antibody 5C6-H4and the light chain variable domain of antibody 5C6-L3.

In some embodiments, the anti-Siglec-9 antibody comprises the heavychain variable domain of antibody 12B12-H1 and the light chain variabledomain of antibody 12B12-L1. In some embodiments, the anti-Siglec-9antibody comprises the heavy chain variable domain of antibody 12B12-H2and the light chain variable domain of antibody 12B 12-L1. In someembodiments, the anti-Siglec-9 antibody comprises the heavy chainvariable domain of antibody 12B 12-H2 and the light chain variabledomain of antibody 12B12-L2. In some embodiments, the anti-Siglec-9antibody comprises the heavy chain variable domain of antibody 12B 12-H2and the light chain variable domain of antibody 12B 12-L3.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 2D4. In some embodiments, the anti-Siglec-9 antibodyis an isolated antibody which binds essentially the same TREM2 epitopeas 2D4. In some embodiments, the anti-Siglec-9 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domain of monoclonal antibody 2D4. In some embodiments, theanti-Siglec-9 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domain of monoclonalantibody 2D4. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domain of monoclonal antibody 2D4.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 2D5. In some embodiments, the anti-Siglec-9 antibodyis an isolated antibody which binds essentially the same TREM2 epitopeas 2D5. In some embodiments, the anti-Siglec-9 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domain of monoclonal antibody 2D5. In some embodiments, theanti-Siglec-9 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domain of monoclonalantibody 2D5. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domain of monoclonal antibody 2D5.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 5B 1. In some embodiments, the anti-Siglec-9antibody is an isolated antibody which binds essentially the same TREM2epitope as 5B1. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain of monoclonal antibody 5B 1. In some embodiments,the anti-Siglec-9 antibody is an isolated antibody comprising theHVR-L1, HVR-L2, and HVR-L3 of the light chain variable domain ofmonoclonal antibody 5B 1. In some embodiments, the anti-Siglec-9antibody is an isolated antibody comprising the HVR-H1, HVR-H2, andHVR-H3 of the heavy chain variable domain and the HVR-L1, HVR-L2, andHVR-L3 of the light chain variable domain of monoclonal antibody 5B1.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 6B2. In some embodiments, the anti-Siglec-9 antibodyis an isolated antibody which hinds essentially the same TREM2 epitopeas 6B2. In some embodiments, the anti-Siglec-9 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domain of monoclonal antibody 6B2. In some embodiments, theanti-Siglec-9 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domain of monoclonalantibody 6B2. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domain of monoclonal antibody 6B2.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 6D8. In some embodiments, the anti-Siglec-9 antibodyis an isolated antibody which binds essentially the same TREM2 epitopeas 6D8. In some embodiments, the anti-Siglec-9 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domain of monoclonal antibody 6D8. In some embodiments, theanti-Siglec-9 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domain of monoclonalantibody 6D8. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domain of monoclonal antibody 6D8.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 7H12. In some embodiments, the anti-Siglec-9antibody is an isolated antibody which binds essentially the same TREM2epitope as 7H12. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain of monoclonal antibody 7H12. In some embodiments,the anti-Siglec-9 antibody is an isolated antibody comprising theHVR-L1, HVR-L2, and HVR-L3 of the light chain variable domain ofmonoclonal antibody 7H12. In some embodiments, the anti-Siglec-9antibody is an isolated antibody comprising the HVR-H1, HVR-H2, andHVR-H3 of the heavy chain variable domain and the HVR-L1, HVR-L2, andHVR-L3 of the light chain variable domain of monoclonal antibody 7H12.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 5C6. In some embodiments, the anti-Siglec-9 antibodyis an isolated antibody which binds essentially the same TREM2 epitopeas 5C6. In some embodiments, the anti-Siglec-9 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domain of monoclonal antibody 5C6. In some embodiments, theanti-Siglec-9 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domain of monoclonalantibody 5C6. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domain of monoclonal antibody 5C6.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 12B 12. In some embodiments, the anti-Siglec-9antibody is an isolated antibody which binds essentially the same TREM2epitope as 12B12. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain of monoclonal antibody 12B 12. In someembodiments, the anti-Siglec-9 antibody is an isolated antibodycomprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variabledomain of monoclonal antibody 12B 12. In some embodiments, theanti-Siglec-9 antibody is an isolated antibody comprising the HVR-H1,HVR-H2, and HVR-H3 of the heavy chain variable domain and the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domain of monoclonalantibody 12B12.

In some embodiments, the anti-Siglec-9 antibody is anti-Siglec-9monoclonal antibody 17C2. In some embodiments, the anti-Siglec-9antibody is an isolated antibody which binds essentially the same TREM2epitope as 17C2. In some embodiments, the anti-Siglec-9 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domain of monoclonal antibody 17C2. In some embodiments,the anti-Siglec-9 antibody is an isolated antibody comprising theHVR-L1, HVR-L2, and HVR-L3 of the light chain variable domain ofmonoclonal antibody 17C2. In some embodiments, the anti-Siglec-9antibody is an isolated antibody comprising the HVR-H1, HVR-H2, andHVR-H3 of the heavy chain variable domain and the HVR-L1, HVR-L2, andHVR-L3 of the light chain variable domain of monoclonal antibody 17C2.

Any of the antibodies of the present disclosure may be produced by acell line. In some embodiments, the cell line may be a mammalian cellline. In certain embodiments, the cell line may be a hybridoma cellline. In other embodiments, the cell line may be a yeast cell line. Anycell line known in the art suitable for antibody production may be usedto produce an antibody of the present disclosure. Exemplary cell linesfor antibody production are described throughout the present disclosure.

In some embodiments, the anti-Siglec-9 antibody is an anti-Siglec-9monoclonal antibody selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6,12B12, and 17C2. In certain embodiments, the anti-Siglec-9 antibody isan antagonist antibody. In certain embodiments, the anti-Siglec-9antibody is an agonist antibody or an inert antibody.

Anti-Siglec-9 Antibody Binding Affinity

The dissociation constants (K_(D)) of anti-Siglec-9 antibodies for humanSiglec-9, mammalian Siglec-9, or both, may be less than 100 nM, lessthan 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10nM, less than 9.5 nM, less than 9 nM, less than 8.5 nM, less than 8 nM,less than 7.5 nM, less than 7 nM, less than 6.9 nM, less than 6.8 nM,less than 6.7 nM, less than 6.6 nM, less than 6.5 nM, less than 6.4 nM,less than 6.3 nM, less than 6.2 nM, less than 6.1 nM, less than 6 nM,less than 5.9 nM, less than 5.8 nM, less than 5.7 nM, less than 5.6 nM,less than 5.5 nM, less than 5.4 nM, less than 5.3 nM, less than 5.2 nM,less than 5.1 nM, less than 5 nM, less than 4.5 nM, less than 4 nM, lessthan 3.5 nM, less than 3 nM, less than 2.9 nM, less than 2.8 nM, lessthan 2.7 nM, less than 2.6 nM, less than 2.5 nM, less than 2.4 nM, lessthan 2.3 nM, less than 2.2 nM, less than 2.1 nM, less than 2 nM, lessthan 1.5 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, lessthan 0.79 nM, less than 0.78 nM, less than 0.77 nM, less than 0.76 nM,less than 0.75 nM, less than 0.74 nM, less than 0.73 nM, less than 0.72nM, less than 0.71 nM, less than 0.70 nM, less than 0.6 nM, less than0.59 nM, less than 0.58 nM, less than 0.57 nM, less than 0.56 nM, lessthan 0.55 nM, less than 0.54 nM, less than 0.53 nM, less than 0.52 nM,less than 0.51 nM, less than 0.50 nM, less than 0.4 nM, less than 0.3nM, less than 0.29 nM, less than 0.28 nM, less than 0.27 nM, less than0.26 nM, less than 0.25 nM, less than 0.24 nM, less than 0.23 nM, lessthan 0.22 nM, less than 0.21 nM, less than 0.2 nM, less than 0.1 nM, orless than 0.05 nM (i.e., 50 pM). In some embodiments, the antibody has adissociation constant (K_(D)) for human Siglec-9, mammalian Siglec-9, orboth, that ranges from less than 10 nM to less than 10 pM (i.e., 0.01nM). In some embodiments, the antibody has a dissociation constant(K_(D)) for human Siglec-9 that ranges from about 9 nM to about 300 pM,or less than 300 pM. In some embodiments, the antibody has adissociation constant (K_(D)) for human Siglec-9 that ranges from about9 nM to about 230 pM, or less than 2300 pM. Dissociation constants maybe determined through any analytical technique, including anybiochemical or biophysical technique such as ELISA, surface plasmonresonance (SPR), bio-layer interferometry (see, e.g., Octet System byForteBio), isothermal titration calorimetry (ITC), differential scanningcalorimetry (DSC), circular dichroism (CD), stopped-flow analysis, andcolorimetric or fluorescent protein melting analyses. In someembodiments, the K_(D) is determined using a full-length antibody in amonovalent form. In some embodiments, the K_(D) is determined utilizing,for example, a surface plasmon resonance assay or Fortebio assay asdescribed herein (see, e.g., Example 1).

Additional anti-Siglec-9 antibodies, e.g., antibodies that specificallybind to a Siglec-9 protein of the present disclosure, may be identified,screened, and/or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

Anti-Siglec-9 Antibodies Capable of Binding Fc Gamma Receptors

In some embodiments, anti-Siglec-9 antibodies of the present disclosureretain the ability to bind Fc gamma receptors. In some embodiments, suchantibodies when they have the correct epitope specificity that iscompatible with receptor activation may have features that enable themto cluster and transiently stimulate, for example, the Siglec-9receptor. In some embodiments, such antibodies may subsequently act aslonger-term inhibitors of Siglec-9 expression and/or one or moreactivities of a Siglec-9 protein by inducing Siglec-9 degradation,Siglec-9 desensitization, Siglec-9 cleavage, Siglec-9 internalization,Siglec-9 shedding, downregulation of Siglec-9 expression, and/orlysosomal degradation of Siglec-9.

In vivo, anti-Siglec-9 antibodies of the present disclosure may clusterreceptors and transiently activate Siglec-9 by any one or more ofmultiple potential mechanisms. Some isotypes of human antibodies such asIgG2 have, due to their unique structure, an intrinsic ability tocluster receptors, or retain receptors in a clustered configuration,thereby transiently activating receptors such as Siglec-9 withoutbinding to an Fc receptor (e.g., White et al., (2015) Cancer Cell 27,138-148).

In some embodiments, other antibodies may cluster receptors (e.g.,Siglec-9) by binding to Fcg receptors on adjacent cells. In someembodiments, binding of the constant IgG Fc region of the antibody toFcg receptors may lead to aggregation of the antibodies, and theantibodies in turn may aggregate the receptors to which they bindthrough their variable region (Chu et al (2008) Mol Immunol,45:3926-3933; and Wilson et al., (2011) Cancer Cell 19, 101-113). Insome embodiments, binding to the inhibitory Fcg receptor FcgR (FcgRIIB)that does not elicit cytokine secretion, oxidative burst, increasedphagocytosis, and enhanced antibody-dependent, cell-mediatedcytotoxicity (ADCC) is a preferred way to cluster antibodies in vivo,since binding to FcgRIIB is not associated with adverse immune responseeffects.

There are other mechanisms by which anti-Siglec-9 antibodies of thepresent disclosure can cluster receptors. For example, antibodyfragments (e.g., Fab fragments) that are cross-linked together may beused to cluster receptors (e.g., Siglec-9) in a manner similar toantibodies with Fc regions that bind Fcg receptors, as described above.In some embodiments, cross-linked antibody fragments (e.g., Fabfragments) may transiently function as agonist antibodies if they inducereceptor clustering on the cell surface and bind an appropriate epitopeon the target (e.g., Siglec-9).

Therefore, in some embodiments, antibodies of the present disclosurethat bind a Siglec-9 protein may include agonist antibodies that due totheir epitope specificity bind Siglec-9 and transiently activate one ormore Siglec-9 activities before they, for example, decrease cellularlevels of Siglec-9, inhibit one or more Siglec-9 activities (e.g., dueto decreased cellular levels of Siglec-9), and/or inhibit interaction(e.g., binding) between Siglec-9 and one or more Siglec-9 ligands. Insome embodiments, such antibodies may bind to the ligand-binding site onSiglec-9 and transiently mimic the action of a natural ligand.Alternatively, such antibodies may stimulate the target antigen totransduce signal by binding to one or more domains that are not theligand-binding sites. In some embodiments, such antibodies would notinterfere with ligand binding. In some embodiments, regardless ofwhether antibodies bind or do not bind to the ligand-binding site onSiglec-9, the antibodies may subsequently act as longer term inhibitorsof Siglec-9 expression and/or one or more activities of a Siglec-9protein by inducing Siglec-9 degradation, Siglec-9 desensitization,Siglec-9 cleavage, Siglec-9 internalization, Siglec-9 shedding,downregulation of Siglec-9 expression, and/or lysosomal degradation ofSiglec-9.

In some embodiments, an anti-Siglec-9 antibody of the present disclosureis a transient agonist antibody that transiently induces one or moreactivities of a Siglec-9 protein. In some embodiments, the antibodytransiently induces the one or more activities after binding to aSiglec-9 protein that is expressed in a cell. In some embodiments, theSiglec-9 protein is expressed on a cell surface. In some embodiments,the one or more activities of a Siglec-9 protein that are transientlyinduced by transient agonist anti-Siglec-9 antibodies of the presentdisclosure may include, without limitation, phosphorylation of Tyr-433and Tyr-456 by a Src family tyrosine kinase, such as Syk, LCK, FYM,and/or ZAP70; recruitment of and binding to the tyrosine-specificprotein phosphatases SHP1 and SHP2; recruitment of and binding toPLC-gamma1, which acts as a guanine nucleotide exchange factor forDynamini-1; recruitment of and binding to SH2-domain containing protein(e.g., Crk1); recruitment of and binding to the spleen tyrosine kinaseSyk; recruitment of and binding to SH3-SH2-SH3 growth factorreceptor-bound protein 2 (Grb2); recruitment of and binding to multipleSH2-containing proteins; modulated expression of one or morepro-inflammatory cytokines, such as FN-α4, IFN-beta, IL-1β, IL-1alpha,TNF-α, IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ, OSM, CNTF,GM-CSF, IL-11, IL-12, IL-17, IL-18, CRP, MCP-1, and MIP-1-beta;modulated expression of one or more pro-inflammatory cytokines in one ormore cells selected from macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, monocytes, osteoclasts, Tcells, T helper cells, cytotoxic T cells, granulocytes, and microglialcells; increased expression of one or more anti-inflammatory cytokines,such as IL-4, IL-10, IL-13, IL-35, IL-16, TGF-beta, IL-1Rα, G-CSF, andsoluble receptors for TNF, IFN-beta1a, IFN-beta1b, or IL-6; modulatedexpression of one or more anti-inflammatory cytokines in one or morecells selected from macrophages, neutrophils, NK cells, dendritic cells,bone marrow-derived dendritic cells, monocytes, osteoclasts, T cells, Thelper cells, cytotoxic T cells, granulocytes, and microglial cells;modulate expression of one or more proteins selected from C1qa, C1qB,C1qC, C1s, C1R, C4, C2, C3, ITGB2, HMOX1, LAT2, CASP1, CSTA, VSIG4,MS4A4A, C3AR1, GPX1, TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX, andPYCARD; inhibition of extracellular signal-regulated kinase (ERK)phosphorylation; decreasing tyrosine phosphorylation on one or morecellular proteins, optionally, wherein the one or more cellular proteinscomprise ZAP-70 and the tyrosine phosphorylation occurs on Tyr-319 ofZAP-70; modulated expression of C—C chemokine receptor 7 (CCR7);inhibition of microglial cell chemotaxis toward CCL19-expressing andCCL21-expressing cells; decreasing T cell proliferation induced by oneor more cells selected from dendritic cells, bone marrow-deriveddendritic cells, monocytes, microglia, M1 microglia, activated M1microglia, M2 microglia, macrophages, neutrophils, NK cells, M1macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages,activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2neutrophils, and M2 NK cells; inhibition of osteoclast production,decreased rate of osteoclastogenesis, or both; decreasing survival ofone or more cells selected from dendritic cells, bone marrow-deriveddendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; decreasing proliferation of one or morecells selected from dendritic cells, bone marrow-derived dendriticcells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; inhibiting migration of one or more cellsselected from dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibiting one or more functions of one or more cellsselected from dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibiting maturation of one or more cells selected fromdendritic cells, bone marrow-derived dendritic cells, macrophages,neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells,activated M1 macrophages, activated M1 neutrophils, activated M1 NKcells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; inhibition of one or more types of clearance selected fromapoptotic neuron clearance, nerve tissue debris clearance, dysfunctionalsynapse clearance, non-nerve tissue debris clearance, bacteriaclearance, other foreign body clearance, disease-causing proteinclearance, disease-causing peptide clearance, and tumor cell clearance;optionally wherein the disease-causing protein is selected from amyloidbeta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursorprotein or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUSprotein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein,prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase,ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10,Lewy body, atrial natriuretic factor, islet amyloid polypeptide,insulin, apolipoprotein AI, serum amyloid A, medin, prolactin,transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides and the tumor cell is from a cancer selected frombladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer; inhibition of phagocytosis of one or more of apoptoticneurons, nerve tissue debris, dysfunctional synapses, non-nerve tissuedebris, bacteria, other foreign bodies, disease-causing proteins,disease-causing peptides, disease-causing nucleic acids, or tumor cells;optionally wherein the disease-causing nucleic acids are antisenseGGCCCC (G2C4) repeat-expansion RNA, the disease-causing proteins areselected from amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AT, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides, and the tumor cells are from a cancer selectedfrom bladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, orthyroid cancer; binding to Siglec-9 ligand on tumor cells; binding toSiglec-9 ligand on cells selected from neutrophils, dendritic cells,bone marrow-derived dendritic cells, monocytes, microglia, macrophages,and NK cells; inhibition of tumor cell killing by one or more ofmicroglia, macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, neutrophils, T cells, T helper cells, orcytotoxic T cells; inhibiting anti-tumor cell proliferation activity ofone or more of microglia, macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, neutrophils, T cells, Thelper cells, or cytotoxic T cells; inhibition of anti-tumor cellmetastasis activity of one or more of microglia, macrophages,neutrophils, NK cells, dendritic cells, bone marrow-derived dendriticcells, neutrophils, T cells, T helper cells, or cytotoxic T cells;inhibition of one or more ITAM motif containing receptors, optionallywherein the one or more ITAM motif containing receptors are selectedfrom TREM1, TREM2, Sirp beta, FcgR, DAP10, and DAP12; inhibition ofsignaling by one or more pattern recognition receptors (PRRs),optionally wherein the one or more PRRs are selected from receptors thatidentify pathogen-associated molecular patterns (PAMPs), receptors thatidentify damage-associated molecular patterns (DAMPs), and anycombination thereof; inhibition of one or more receptors comprising themotif D/Ex₀ ₂YxxL/IX₆ ₈YxxL/i (SEQ ID NO: 252); inhibition of signalingby one or more Toll-like receptors; inhibition of the JAK-STAT signalingpathway; inhibition of nuclear factor kappa-light-chain-enhancer ofactivated B cells (NFκB); de-phosphorylation of an ITAM motif containingreceptor; modulated expression of one or more inflammatory receptors,proteins of the complement cascade, and/or receptors that are expressedon immune cells, optionally wherein the one or more inflammatoryreceptors, proteins of the complement cascade, and/or receptors that areexpressed on immune cells comprise CD86, C1qa, C1qB, C1qC, C1s, C1R, C4,C2, C3, ITGB2, HMOX1, LAT2, CASP1, CSTA, VSIG4, MS4A4A, C3AR1, GPX1,TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX, and/or PYCARD, and the oneor more inflammatory receptors, proteins of the complement cascade,and/or receptors that are expressed on immune cells are expressed on oneor more of microglia, macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, neutrophils, T cells, Thelper cells, or cytotoxic T cells; increasing expression of one or moreSiglec-9-dependent genes; normalization of disrupted Siglec-9-dependentgene expression; decreasing expression of one or more ITAM-dependentgenes, optionally wherein the one more ITAM-dependent genes areactivated by nuclear factor of activated T cells (NFAT) transcriptionfactors; promoting or rescuing functionality of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, and regulatory T cells;increasing infiltration of one or more of immunosuppressor dendriticcells, immunosuppressor macrophages, immunosuppressor neutrophils,immunosuppressor NK cells, myeloid-derived suppressor cells,tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, and regulatory T cells into tumors;increasing the number of tumor-promoting myeloid/granulocyticimmune-suppressive cells in a tumor, in peripheral blood, or otherlymphoid organ; enhancing tumor-promoting activity of myeloid-derivedsuppressor cells; increasing expression of tumor-promoting cytokines ina tumor or in peripheral blood, optionally wherein the tumor-promotingcytokines are TGF-beta or IL-10; increasing tumor infiltration oftumor-promoting FoxP3+ regulatory T lymphocytes; enhancingtumor-promoting activity of myeloid-derived suppressor cells (MDSC);decreasing activation of tumor-specific T lymphocytes with tumor killingpotential; decreasing infiltration of tumor-specific NK cells with tumorkilling potential; decreasing the tumor killing potential of NK cells;decreasing infiltration of tumor-specific B lymphocytes with potentialto enhance immune response; decreasing infiltration of tumor-specific Tlymphocytes with tumor killing potential; increasing tumor volume;increasing tumor growth rate; increasing metastasis; increasing rate oftumor recurrence; decreasing efficacy of one or more immune-therapiesthat modulate anti-tumor T cell responses, optionally wherein the one ormore immune-therapies are immune-therapies that target one or moretarget proteins selected from PD1/PDL1, CD40, OX40, ICOS, CD28,CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM,LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR,LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1 receptor, and anycombination thereof, or of one or more cancer vaccines; inhibition ofPLCγ/PKC/calcium mobilization; and inhibition of PI3K/Akt, Ras/MAPKsignaling. Anti-Siglec-9 antibodies of the present disclosure may betested for their ability to transiently induce one or more activities ofa Siglec-9 protein utilizing any suitable technique or assay known inthe art and disclosed herein. Regardless of the activities that suchantibodies transiently induce, such antibodies may subsequently act aslonger-term inhibitors of Siglec-9 expression and/or one or moreactivities of a Siglec-9 protein by inducing Siglec-9 degradation,Siglec-9 desensitization, Siglec-9 cleavage, Siglec-9 internalization,Siglec-9 shedding, downregulation of Siglec-9 expression, and/orlysosomal degradation of Siglec-9. In some embodiments, the Siglec-9antibody transiently induces one or more activities of a Siglec-9protein independently of binding to an Fc receptor.

Exemplary antibody Fc isotypes and modifications are provided in Table Cbelow. In some embodiments, an anti-Siglec-9 antibody of the presentdisclosure that is capable of binding an Fc gamma receptor has an Fcisotype listed in Table C below.

TABLE C Exemplary anti-Siglec-9 antibody Fc isotypes that are capable ofbinding Fc gamma receptor Fc Isotype Mutation (EU or Kabat numberingscheme) IgG1 N297A IgG1 D265A and N297A IgG1 D270A IgG1 L234A and L235AL234A and G237A L234A and L235A and G237A IgG1 D270A, and/or P238D,and/or L328E, and/or E233D, and/or G237D, and/or H268D, and/or P271G,and/or A330R IgG1 P238D and L328E and E233D and G237D and H268D andP271G and A330R IgG1 P238D and L328E and G237D and H268D and P271G andA330R IgG1 P238D and S267E and L328F and E233D and G237D and H268D andP271G and A330R IgG1 P238D and S267E and L328F and G237D and H268D andP271G and A330R IgG2 V234A and G237A IgG4 L235A and G237A and E318A IgG4S228P and L236E IgG2/4 hybrid IgG2 aa 118 to 260 and IgG4 aa 261 to 447H268Q and V309L; and A330S and P331S IgG1 C226S and C229S and E233P andL234V and L235A IgG1 L234F and L235E and P331S IgG2 C232S or C233S IgG2A330S and P331S IgG1 S267E, and L328F S267E alone IgG2 S267E and L328FIgG4 S267E and L328F IgG2 WT HC with Kappa (light chain) LC HC C127Swith Kappa LC Kappa LC C214S Kappa LC C214S and HC C233S Kappa LC C214Sand HC C232S Any of the above listed mutations together with P330S andP331S mutations F(ab’)2 fragment of WT IgG1 and any of the above listedmutations IgG1 Substitute the Constant Heavy 1 (CH1) and hinge region ofIgG1 With CH1 and hinge region of IGg2 ASTKGPSVFP LAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPSNTKVDKTVER KCCVECPPCP (SEQ ID NO: 171) With a Kappa LC IgG1 Any of theabove listed mutations together with A330L and/or L234F and/or L235Eand/or P331S IgGl, IgG2, or IgG4 Any of the above listed mutationstogether with M252Y and/or S254T and/or T256E Mouse IgG1 For mousedisease models IgG4 WT

In addition to the isotypes described in Table C, and without wishing tobe bound to theory, it is thought that antibodies with human IgG1 orIgG3 isotypes and mutants thereof (e.g. Strohl (2009) Current Opinion inBiotechnology 2009, 20:685-691) that bind the Fcg Receptors I, IIA, IIC,IIIA, IIIB in human and/or Fcg Receptors I, III and IV in mouse, mayalso act as transient agonist antibodies.

In some embodiments, the Fc gamma receptor-binding antibody is of theIgG class, the IgM class, or the IgA class. In some embodiments, the Fcgamma receptor-binding antibody has an IgG1, IgG2, IgG3, or IgG4isotype.

In certain embodiments, the Fc gamma receptor-binding antibody has anIgG2 isotype. In some embodiments, the Fc gamma receptor-bindingantibody contains a human IgG2 constant region. In some embodiments, thehuman IgG2 constant region includes an Fc region. In some embodiments,the Fc gamma receptor-binding antibody binds an inhibitory Fc receptor.In certain embodiments, the inhibitory Fc receptor is inhibitoryFc-gamma receptor IIB (FcγIIB). In some embodiments, the Fc regioncontains one or more modifications. For example, in some embodiments,the Fc region contains one or more amino acid substitutions (e.g.,relative to a wild-type Fc region of the same isotype). In someembodiments, the one or more amino acid substitutions are selected fromV234A (Alegre et al., (1994) Transplantation 57:1537-1543. 31; Xu etal., (2000) Cell Immunol, 200:16-26), G237A (Cole et al. (1999)Transplantation, 68:563-571), H268Q, V309L, A330S, P331S (US2007/0148167; Armour et al. (1999) Eur J Immunol 29: 2613-2624; Armouret al. (2000) The Haematology Journal 1(Suppl.1):27; Armour et al.(2000) The Haematology Journal 1(Suppl.1):27), C232S, and/or C233S(White et al. (2015) Cancer Cell 27, 138-148), S267E, L328F (Chu et al.,(2008) Mol Immunol, 45:3926-3933), M252Y, S254T, and/or T256E, where theamino acid position is according to the EU or Kabat numberingconvention.

In some embodiments, the Fc gamma receptor-binding antibody has an IgG2isotype with a heavy chain constant domain that contains a C127S aminoacid substitution, where the amino acid position is according to the EUor Kabat numbering convention (White et al., (2015) Cancer Cell 27,138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; andWO2008079246).

In some embodiments, the Fc gamma receptor-binding antibody has an IgG2isotype with a Kappa light chain constant domain that contains a C214Samino acid substitution, where the amino acid position is according tothe EU or Kabat numbering convention (White et al., (2015) Cancer Cell27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; andWO2008079246).

In certain embodiments, the Fc gamma receptor-binding antibody has anIgG1 isotype. In some embodiments, the Fc gamma receptor-bindingantibody contains a mouse IgG1 constant region. In some embodiments, theFc gamma receptor-binding antibody contains a human IgG1 constantregion. In some embodiments, the human IgG1 constant region includes anFc region. In some embodiments, the Fc gamma receptor-binding antibodybinds an inhibitory Fc receptor. In certain embodiments, the inhibitoryFc receptor is inhibitory Fc-gamma receptor IIB (FcγIIB). In someembodiments, the Fc region contains one or more modifications. Forexample, in some embodiments, the Fc region contains one or more aminoacid substitutions (e.g., relative to a wild-type Fc region of the sameisotype). In some embodiments, the one or more amino acid substitutionsare selected from N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411),D265A (Shields et al. (2001) R. J. Biol. Chem. 276, 6591-6604), D270A,L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA,92:11980-11984; Alegre et al., (1994) Transplantation 57:1537-1543. 31;Xu et al., (2000) Cell Immunol, 200:16-26), G237A (Alegre et al. (1994)Transplantation 57:1537-1543. 31; Xu et al. (2000) Cell Immunol,200:16-26), P238D, L328E, E233D, G237D, H268D, P271G, A330R, C226S,C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood,109:1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA2008, 105:20167-20172), S267E, L328F, A330L, M252Y, S254T, T256E, N297Q,P238S, P238A, A327Q, A327G, P329A, K322A, and/or T394D, where the aminoacid position is according to the EU or Kahat numbering convention.

In some embodiments, the antibody includes an IgG2 isotype heavy chainconstant domain 1(CH1) and hinge region (White et al., (2015) CancerCell 27, 138-148). In certain embodiments, the IgG2 isotype CH1 andhinge region contain the amino acid sequence ofASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 171). Insome embodiments, the antibody Fc region contains a S267E amino acidsubstitution, a L328F amino acid substitution, or both, and/or a N297Aor N297Q amino acid substitution, where the amino acid position isaccording to the EU or Kabat numbering convention.

In certain embodiments, the Fc gamma receptor-binding antibody has anIgG4 isotype. In some embodiments, the Fc gamma receptor-bindingantibody contains a human IgG4 constant region. In some embodiments, thehuman IgG4 constant region includes an Fc region. In some embodiments,the Fc gamma receptor-binding antibody binds an inhibitory Fc receptor.In certain embodiments, the inhibitory Fc receptor is inhibitoryFc-gamma receptor ITB (FcγIIB). In some embodiments, the Fc regioncontains one or more modifications. For example, in some embodiments,the Fc region contains one or more amino acid substitutions (e.g.,relative to a wild-type Fc region of the same isotype). In someembodiments, the one or more amino acid substitutions are selected fromL235A, G237A, S228P, L236E (Reddy et al., (2000) J Immunol,164:1925-1933), S267E, E318A, L328F, M252Y, S254T, and/or T256E, wherethe amino acid position is according to the EU or Kabat numberingconvention.

In certain embodiments, the Fc gamma receptor-binding antibody has ahybrid IgG2/4 isotype. In some embodiments, the Fc gammareceptor-binding antibody includes an amino acid sequence containingamino acids 118 to 260 according to EU or, Kabat numbering of human IgG2and amino acids 261-447 according to EU or, Kabat numbering of humanIgG4 (WO 1997/11971; WO 2007/106585).

In certain embodiments, the antibody contains a mouse IgG4 constantregion (Bartholomaeus, et al. (2014). J. Immunol. 192, 2091-2098).

In some embodiments, the Fc region further contains one or moreadditional amino acid substitutions selected from A330L, L234F; L235E,or P331S according to EU or, Kahat numbering; and any combinationthereof.

Inert Antibodies

Another class of anti-Siglec-9 antibodies of the present disclosureincludes inert antibodies. As used herein, “inert” antibodies refer toantibodies that specifically bind their target antigen (e.g., Siglec-9)but do not modulate (e.g., decrease/inhibit or activate/induce) antigenfunction. For example, in the case of Siglec-9, inert antibodies do notmodulate cellular levels of Siglec-9, do not modulate interaction (e.g.,binding) between Siglec-9 and one or more Siglec-9 ligands, or do notmodulate one or more activities of a Siglec-9 protein. In someembodiments, antibodies that do not have the ability to cluster Siglec-9on the cell surface may be inert antibodies even if they have an epitopespecificity that is compatible with receptor activation.

In some embodiments, antibodies that bind a Siglec-9 protein may includeantibodies that bind Siglec-9 but, due to their epitope specificity, orcharacteristics, do not decrease cellular levels of Siglec-9 and/orinhibit interaction (e.g., binding) between Siglec-9 and one or moreSiglec-9 ligands. In some embodiments, such antibodies can be used ascargo to, for example, transport toxins (e.g., chemotherapeutics) intotumor cells. Such antibodies may be superior to current commerciallyavailable anti-Siglec-9 antibodies that reduce cellular levels ofSiglec-9, such as gemtuzumab zogamicin, which is conjugated to acytotoxic agent from the class of calicheamicins and is used to targetand kill acute myelogenous leukemia tumors (Naito et al., (2000),Leukemia, 14, 1436-1443; Ricart (2011) Clin Cancer Res 17; 6417-6436;Hamann et al., (2002) Journal: Bioconjugate Chemistry, 13, 47-58; Beitzet al., (2001) Clin Cancer Res 7; 1490-6; and Malik M. et al. (2015)Human Molecular Genetics, 1-14.). In some embodiments, inertanti-Siglec-9 antibodies of the present disclosure may be superior tocommercial antibodies, such as gemtuzumab zogamicin, because antibodiesthat do not decrease cellular levels of Siglec-9 will leave Siglec-9intact on the surface of tumor cells for targeting by additionaltoxin-conjugated antibodies. In contrast, antibodies that decreasecellular levels of Siglec-9 will remove Siglec-9 from the cell surfaceand will lead to protection of the tumor cells from further targeting bytoxin-conjugated antibodies. Therefore, in some embodiments, antibodiesof the present disclosure are inert antibodies that bind Siglec-9 butare incapable of decreasing cellular levels of Siglec-9, inhibitinginteraction (e.g., binding) between Siglec-9 and one or more Siglec-9ligands, or inducing one or more activities of a Siglec-9 protein.

Antibodies that either decrease or do not decrease cellular levels ofSiglec-9 on cells can be combined with an inert Fc region that displaysreduced binding to one or more Fcg Receptor. Examples of such Fc regionsand modifications are provided in Table D below. In some embodiments,the antibody with an inert Fc region has an Fc isotype listed in Table Dbelow.

Antagonist Anti-Siglec-9 Antibodies

A third class of anti-Siglec-9 antibodies of the present disclosureincludes antagonist antibodies. In some embodiments, antibodies thatbind a Siglec-9 protein may include antagonist antibodies that reducecellular levels of Siglec-9, inhibit interaction (e.g., binding) betweenSiglec-9 and/or one or more Siglec-9 ligands, and inhibit one or moreactivities of a Siglec-9 protein. Such antibodies inhibit one or moreactivities of a Siglec-9 protein either by preventing interaction (e.g.,binding) between Siglec-9 and one or more Siglec-9 ligands or bypreventing signal transduction from the extracellular domain of Siglec-9into the cell cytoplasm in the presence of one or more Siglec-9 ligands.Antagonist antibodies also can inhibit one or more activities of aSiglec-9 protein by decreasing cell surface levels of Siglec-9 byinducing Siglec-9 degradation, Siglec-9 desensitization, Siglec-9cleavage, Siglec-9 internalization, Siglec-9 shedding, downregulation ofSiglec-9 expression, and/or lysosomal degradation of Siglec-9. In someembodiments, such antagonist anti-Siglec-9 antibodies may nottransiently activate Siglec-9.

In some embodiments, antagonist anti-Siglec-9 antibodies of the presentdisclosure may have the epitope specificity of a transient agonistanti-Siglec-9 antibody of the present disclosure, but have an Fc domainthat is not capable of binding Fcg receptors and thus is unable to, forexample, transiently clustering and activating Siglec-9.

In some embodiments, antagonist anti-Siglec-9 antibodies of the presentdisclosure have, without limitation, one or more of the followingactivities: the ability to decrease binding of a Siglec-9 protein to oneor more Siglec-9 ligands, such as sialic acid-containing glycolipids orsialic acid-containing glycoproteins, the ability to decrease thebinding of a suppressor of cytokine signaling (SOCS) protein (e.g.,SOCS3 protein) to a Siglec-9 protein, the ability to increase theproteasomal degradation of a Siglec-9 protein, the ability to reducefunctional expression of Siglec-9 on the surface of circulatingdendritic cells, macrophages, monocytes, T cells, and/or microglia, theability to decrease or inhibit phosphorylation of Tyr-433 and Tyr-456 bya Src family tyrosine kinase, such as Syk, LCK, FYM, and/or ZAP70; theability to inhibit recruitment of and binding to the tyrosine-specificprotein phosphatases SHP1 and SHP2; the ability to inhibit recruitmentof and binding to PLC-gamma1, which acts as a guanine nucleotideexchange factor for Dynamini-1; the ability to inhibit recruitment ofand binding to SH2-domain containing protein (e.g., Crk1); the abilityto inhibit recruitment of and binding to the spleen tyrosine kinase Syk;the ability to inhibit recruitment of and binding to SH3-SH2-SH3 growthfactor receptor-bound protein 2 (Grb2); the ability to inhibitrecruitment of and binding to multiple SH2-containing proteins; theability to modulate expression of one or more pro-inflammatorycytokines, optionally wherein the one or more anti-inflammatorycytokines are selected from FN-α4, IFN-beta, IL-1β, IL-1alpha, TNF-α,IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ, OSM, CNTF, GM-CSF,IL-11, IL-12, IL-17, IL-18, IL-33, MCP-1, and MIP-1-beta; the ability tomodulate expression of one or more pro-inflammatory cytokines in one ormore cells selected from macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, monocytes, osteoclasts, Tcells, T helper cells, cytotoxic T cells, granulocytes, and microglialcells; the ability to modulate expression of one or moreanti-inflammatory cytokines, optionally wherein the one or moreanti-inflammatory cytokines are selected from IL-4, IL-10, IL-13, IL-35,IL-16, TGF-beta, IL-1Rα, G-CSF, and soluble receptors for TNF,IFN-beta1a, IFN-beta1b, or IL-6; the ability to modulate expression ofone or more anti-inflammatory cytokines in one or more cells selectedfrom macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, monocytes, osteoclasts, T cells, Thelper cells, cytotoxic T cells, granulocytes, and microglial cells; theability to modulate expression of one or more proteins selected fromC1qa, C1qB, C1qC, C1s, C1R, C4, C2, C3, ITGB2, HMOX1, LAT2, CASP1, CSTA,VSIG4, MS4A4A, C3AR1, GPX1, TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX,and PYCARD; the ability to counteract inhibition of extracellularsignal-regulated kinase (ERK) phosphorylation; the ability to preventdecreased tyrosine phosphorylation on one or more cellular proteins,optionally, wherein the one or more cellular proteins comprise ZAP-70and the tyrosine phosphorylation occurs on Tyr-319 of ZAP-70; theability to modulate expression of C—C chemokine receptor 7 (CCR7); theability to prevent inhibition of microglial cell chemotaxis towardCCL19-expressing and CCL21-expressing cells; the ability to preventdecreasing T cell proliferation induced by one or more cells selectedfrom dendritic cells, bone marrow-derived dendritic cells, monocytes,microglia, M1 microglia, activated M1 microglia, M2 microglia,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, and M2 NK cells; theability to prevent inhibition of osteoclast production, the ability toprevent decreased rate of osteoclastogenesis, or both; the ability toprevent decreased survival of one or more cells selected from dendriticcells, bone marrow-derived dendritic cells, macrophages, neutrophils, NKcells, M1 macrophages, M1 neutrophils, M1 NK cells, activated M1macrophages, activated M1 neutrophils, activated M1 NK cells, M2macrophages, M2 neutrophils, M2 NK cells, monocytes, osteoclasts, Tcells, T helper cells, cytotoxic T cells, granulocytes, neutrophils,microglia, M1 microglia, activated M1 microglia, and M2 microglia; theability to prevent decreased proliferation of one or more cells selectedfrom dendritic cells, bone marrow-derived dendritic cells, macrophages,neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells,activated M1 macrophages, activated M1 neutrophils, activated M1 NKcells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; the ability to enhance migration of one or more cellsselected from dendritic cells, bone marrow-derived dendritic cells,macrophages, neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1NK cells, activated M1 macrophages, activated M1 neutrophils, activatedM1 NK cells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; the ability to prevent a decrease in one or more functions ofone or more cells selected from dendritic cells, bone marrow-deriveddendritic cells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; the ability to enhance maturation of one ormore cells selected from dendritic cells, bone marrow-derived dendriticcells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; the ability to enhance one or more types ofclearance selected from apoptotic neuron clearance, nerve tissue debrisclearance, dysfunctional synapse clearance, non-nerve tissue debrisclearance, bacteria clearance, other foreign body clearance,disease-causing protein clearance, disease-causing peptide clearance,and tumor cell clearance; optionally wherein the disease-causing proteinis selected from amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides and the tumor cell is from a cancer selected frombladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer; inhibition of phagocytosis of one or more of apoptoticneurons, nerve tissue debris, dysfunctional synapses, non-nerve tissuedebris, bacteria, other foreign bodies, disease-causing proteins,disease-causing peptides, disease-causing nucleic acids, or tumor cells;optionally wherein the disease-causing nucleic acids are antisenseGGCCCC (G2C4) repeat-expansion RNA, the disease-causing proteins areselected from amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides, and the tumor cells are from a cancer selectedfrom bladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, orthyroid cancer; binding to Siglec-9 ligand on tumor cells; binding toSiglec-9 ligand on cells selected from neutrophils, dendritic cells,bone marrow-derived dendritic cells, monocytes, microglia, macrophages,and NK cells; inhibition of tumor cell killing by one or more ofmicroglia, macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, neutrophils, T cells, T helper cells, orcytotoxic T cells; activating anti-tumor cell proliferation activity ofone or more of microglia, macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, neutrophils, T cells, Thelper cells, or cytotoxic T cells; the ability to enhance anti-tumorcell metastasis activity of one or more of microglia, macrophages,neutrophils, NK cells, dendritic cells, bone marrow-derived dendriticcells, neutrophils, T cells, T helper cells, or cytotoxic T cells; theability to enhance the activity of one or more ITAM motif containingreceptors, optionally wherein the one or more ITAM motif containingreceptors are selected from TREM1, TREM2, SIRP beta, Fc gamma receptors(FcgR), DAP10, and DAP12; the ability to enhance signaling by one ormore pattern recognition receptors (PRRs), optionally wherein the one ormore PRRs are selected from receptors that identify pathogen-associatedmolecular patterns (PAMPs), receptors that identify damage-associatedmolecular patterns (DAMPs), and any combination thereof; the ability toenhance activity of one or more receptors comprising the motif D/Ex₀₂YxxL/IX₆ ₈YxxL/I (SEQ ID NO: 252); the ability to enhance signaling byone or more Toll-like receptors; the ability to enhance the JAK-STATsignaling pathway; the ability to enhance the activity of nuclear factorkappa-light-chain-enhancer of activated B cells (NFκB); the ability toincrease phosphorylation of an ITAM motif containing receptor; theability to increase expression of one or more inflammatory receptors,proteins of the complement cascade, and/or receptors that are expressedon immune cells, optionally wherein the one or more inflammatoryreceptors, proteins of the complement cascade, and/or receptors that areexpressed on immune cells comprise CD86, C1qa, C1qB, C1qC, C1s, C1R, C4,C2, C3, ITGB2, HMOX1, LAT2, CASP1, CSTA, VSIG4, MS4A4A, C3AR1, GPX1,TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX, and/or PYCARD, and the oneor more inflammatory receptors, proteins of the complement cascade,and/or receptors that are expressed on immune cells are expressed on oneor more of microglia, macrophages, neutrophils, NK cells, dendriticcells, hone marrow-derived dendritic cells, neutrophils, T cells, Thelper cells, or cytotoxic T cells; the ability to decrease expressionof one or more Siglec-9-dependent genes; the ability to enhanceexpression of one or more ITAM-dependent genes, optionally wherein theone more ITAM-dependent genes are activated by nuclear factor ofactivated T cells (NFAT) transcription factors; the ability to decreaseor otherwise inhibit differentiation of one or more of immunosuppressordendritic cells, immunosuppressor macrophages, immunosuppressorneutrophils, immunosuppressor NK cells, myeloid derived suppressorcells, tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, and regulatory T cells; the ability todecrease or otherwise inhibit functionality of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, and regulatory T cells; theability to decrease or otherwise inhibit infiltration of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, and regulatory T cells intotumors; the ability to decrease or otherwise inhibit the number oftumor-promoting myeloid/granulocytic immune-suppressive cells in atumor, in peripheral blood, or other lymphoid organ; the ability todecrease or otherwise inhibit tumor-promoting activity ofmyeloid-derived suppressor cells; the ability to decrease or otherwiseinhibit expression of tumor-promoting cytokines, such as TGF-beta orIL-10, in a tumor or in peripheral blood; the ability to decrease orotherwise inhibit tumor infiltration of tumor-promoting FoxP3+regulatory T lymphocytes; the ability to decrease or otherwise inhibittumor-promoting activity of myeloid-derived suppressor cells (MDSC); theability to increase or otherwise enhance tumor-specific T lymphocyteswith tumor killing potential; the ability to increase or otherwiseenhance infiltration of tumor-specific NK cells with tumor killingpotential; the ability to increase or otherwise enhance the tumorkilling potential of NK cells; the ability to increase or otherwiseenhance infiltration of tumor-specific B lymphocytes with potential toenhance immune response; the ability to increase or otherwise enhanceinfiltration of tumor-specific T lymphocytes with tumor killingpotential; the ability to decrease tumor volume; the ability to decreasetumor growth rate; the ability to decrease or otherwise inhibitmetastasis; the ability to decrease rate of tumor recurrence; theability to increase or otherwise enhance efficacy of one or moreimmune-therapies that modulate anti-tumor T cell responses, optionallywherein the one or more immune-therapies are immune-therapies thattarget one or more target proteins selected from PD1/PDL1, CD40, OX40,ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3,B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3,TIM4, A2AR, LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1receptor, and any combination thereof, or of one or more cancervaccines; the ability to increase or otherwise enhance PLCγ/PKC/calciummobilization; and the ability to increase or otherwise enhance PI3K/Akt,Ras/MAPK signaling.

In some embodiments, antagonist anti-Siglec-9 antibodies of the presentdisclosure have an Fc region that displays reduced binding to one ormore Fcg Receptor. Examples of such Fc regions and modifications areprovided in Table D below. In some embodiments, the antibody has an Feisotype listed in Table D below.

Antibody Fc Isotypes with Reduced Binding to Fc Gamma Receptors

In some embodiments, anti-Siglec-9 antibodies with reduced binding to Fegamma receptors have an Fc isotype listed in Table D below.

TABLE D Exemplary anti-Siglec-9 antibody Fc isotypes with reducedbinding to Fc gamma receptor Fc Isotype Mutation (EU or Kabat numberingscheme) IgG1 N297A or N297Q IgG1 D265A, D270A, and N297A IgG1 L234A andL235A IgG2 V234A and G237A IgG4 F235A and G237A and E318A E233P and/orF234V N297A or N297Q IgG4 S228P and L236E S241P S241P and L248E S228Pand F234A and L235A IgG2 H268Q and V309L and A330S and P331S IgG1 C220Sand C226S and C229S and P238S IgG1 C226S and C229S and E233P and L234V,and L235A IgG1 E233P and L234V and L235A and G236-deleted P238A D265AN297A A327Q or A327G P329A IgG1 K322A and L234A and L235A IgG1 L234FandL235E and P331S IgG1 or IgG4 T394D IgG2 C232S or C233S N297A or N297QIgG2 V234A and G237A and P238S and H268A and V309L and A330S and P331SIgGl, IgG2, or IgG4 delta a, b, c, ab, ac, g modifications IgG1 Any ofthe above listed mutations together with A330L or L234F and/or L235Eand/or P331S IgG1, IgG2, or IgG4 Any of the above listed mutationstogether with M252Y and/or S254T and/or T256E

In certain embodiments, the anti-Siglec-9 antibody has an IgG 1 isotype.In some embodiments, the antibody contains a mouse IgG1 constant region.In some embodiments, the antibody contains a human IgG1 constant region.In some embodiments, the human IgG1 constant region includes an Fcregion. In some embodiments, the Fc region contains one or moremodifications. For example, in some embodiments, the Fc region containsone or more amino acid substitutions (e.g., relative to a wild-type Fcregion of the same isotype).

In some embodiments, the one or more amino acid substitutions areselected from N297A, N297Q (Bolt S et al. (1993) Eur J Immunol23:403-411), D270A, D265A, L234A, L235A (McEarchern et al., (2007)Blood, 109:1185-1192), C226S, C229S (McEarchern et al., (2007) Blood,109:1185-1192), P238S (Davis et al., (2007) J Rheumatol, 34:2204-2210),E233P, L234V (McEarchern et al., (2007) Blood, 109:1185-1192), P238A,A327Q, A327G, P329A (Shields R L. et al., (2001) J Biol Chem.276(9):6591-604), K322A, L234F, L235E (Hezareh, et al., (2001) J Virol75, 12161-12168; Oganesyan et al., (2008). Acta Crystallographica 64,700-704), P331S (Oganesyan et al., (2008) Acta Crystallographica 64,700-704), T394D (Wilkinson et al. (2013) MAbs 5(3): 406-417), A330L,M252Y, S254T, and/or T256E, where the amino acid position is accordingto the EU or Kabat numbering convention. In certain embodiments, the Fcregion further includes an amino acid deletion at a positioncorresponding to glycine 236 according to the EU or Kabat numberingconvention.

In some embodiments, the anti-Siglec-9 antibody has an IgG1 isotype witha heavy chain constant region that contains a C220S amino acidsubstitution according to the EU or Kabat numbering convention. In someembodiments, the Fc region further contains one or more additional aminoacid substitutions selected from t A330L, L234F; L235E, and/or P331Saccording to EU or Kabat numbering convention. In certain embodiments,the anti-Siglec-9 antibody has an IgG2 isotype. In some embodiments, theanti-Siglec-9 antibody contains a human IgG2 constant region. In someembodiments, the human IgG2 constant region includes an Fc region. Insome embodiments, the Fc region contains one or more modifications. Forexample, in some embodiments, the Fc region contains one or more aminoacid substitutions (e.g., relative to a wild-type Fc region of the sameisotype). In some embodiments, the one or more amino acid substitutionsare selected from P238S, V234A, G237A, H268A, H268Q, H268E, V309L,N297A, N297Q, V309L, A330S, P331S, C232S, C233S, M252Y, S254T, and/orT256E, where the amino acid position is according to the EU or Kabatnumbering convention (Vafa O. et al., (2014) Methods 65:114-126).

In certain embodiments, the anti-Siglec-9 antibody has an IgG4 isotype.In some embodiments, the anti-Siglec-9 antibody contains a human IgG4constant region. In some embodiments, the human IgG4 constant regionincludes an Fc region. In some embodiments, the Fc region contains oneor more modifications. For example, in some embodiments, the Fc regioncontains one or more amino acid substitutions (e.g., relative to awild-type Fc region of the same isotype). In some embodiments, the oneor more amino acid substitutions are selected from E233P, F234V, L235A,G237A, E318A (Hutchins et al. (1995) Proc Natl Acad Sci USA,92:11980-11984), S228P, L234A/F234A, L236E, S241P, L248E (Reddy et al.,(2000) J Immunol, 164:1925-1933; Angal et al., (1993) Mol Immunol.30(1):105-8; U.S. Pat. No. 8,614,299 B2; Vafa O. et al., (2014) Methods65:114-126), T394D, M252Y, S254T, T256E, N297A, and/or N297Q, where theamino acid position is according to the EU or Kabat numberingconvention.

In some embodiments, the Fc region further contains one or moreadditional amino acid substitutions selected from a M252Y, S254T, and/orT256E, where the amino acid position is according to the EU or Kabatnumbering convention.

Further IgG Mutations

In some embodiments, one or more of the IgG1 variants described hereinmay be combined with an A330L mutation (Lazar et al., (2006) Proc NatlAcad Sci USA, 103:4005-4010), or one or more of L234F, L235E, and/orP331S mutations (Sazinsky et al., (2008) Proc Natl Acad Sci USA,105:20167-20172), where the amino acid position is according to the EUor Kabat numbering convention, to eliminate complement activation. Insome embodiments, the IgG variants described herein may be combined withone or more mutations to enhance the anti-Siglec-9 antibody half-life inhuman serum (e.g. M252Y, S254T, T256E mutations according to the EU orKabat numbering convention) (Dall'Acqua et al., (2006) J Biol Chem,281:23514-23524; and Strohl e al., (2009) Current Opinion inBiotechnology, 20:685-691).

In some embodiments, an IgG4 variant of the present disclosure may becombined with an S228P mutation according to the EU or Kabat numberingconvention (Angal et al., (1993) Mol Immunol, 30:105-108) and/or withone or more mutations described in Peters et al., (2012) J Biol Chem.13; 287(29):24525-33) to enhance antibody stabilization.

Bispecific Antibodies

Certain aspects of the present disclosure relate to bispecificantibodies that bind to one or more domains on a Siglec-9 protein of thepresent disclosure and a second antigen. Methods of generatingbispecific antibodies are well known in the art and described herein. Insome embodiments, bispecific antibodies of the present disclosure bindto one or more amino acid residues of a Siglec-9 protein of the presentdisclosure, such as one or more amino acid residues of human Siglec-9(SEQ ID NO: 1), or amino acid residues on a Siglec-9 proteincorresponding to amino acid residues of SEQ ID NO: 1. In someembodiments, bispecific antibodies of the present disclosure recognize afirst antigen and a second antigen. In some embodiments, the firstantigen is a Siglec-9 protein or a naturally occurring variant thereof.In some embodiments, the second antigen is also a Siglec-9 protein, or anaturally occurring variant thereof. In some embodiments, the secondantigen is an antigen facilitating transport across theblood-brain-barrier (see, e.g., Gabathuler R., Neurobiol. Dis. 37 (2010)48-57). Such second antigens include, without limitation, transferrinreceptor (TR), insulin receptor (HIR), insulin-like growth factorreceptor (IGFR), low-density lipoprotein receptor related proteins 1 and2 (LPR-1 and 2), diphtheria toxin receptor, CRM 197, a llama singledomain antibody, TMEM 30(A), a protein transduction domain, TAT, Syn-B,penetratin, a poly-arginine peptide, Angiopep peptides such as ANG1005(see, e.g., Gabathuler, 2010), and other cell surface proteins that areenriched on blood-brain barrier endothelial cells (see, e.g., Daneman etal., PLoS One. 2010 Oct. 29; 5(10):e13741). In some embodiments, thesecond antigen is a disease-causing protein including, withoutlimitation, amyloid beta, oligomeric amyloid beta, amyloid beta plaques,amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides. In some embodiments, the second antigen is one ormore ligands and/or proteins expressed on immune cells, includingwithout limitation, PD1/PDL1, CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27,GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30,TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5,CD39, CD73, and phosphatidylserine. In some embodiments, the secondantigen is a protein, lipid, polysaccharide, or glycolipid expressed onone or more tumor cells.

Antibody Fragments

Certain aspects of the present disclosure relate to antibody fragmentsthat bind to one or more of a Siglec-9 protein of the presentdisclosure, a naturally occurring variant of a Siglec-9 protein, and adisease variant of a Siglec-9 protein. In some embodiments, the antibodyfragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.

In some embodiments, the antibody fragment is used in combination with asecond Siglec-9 antibody and/or with one or more antibodies thatspecifically bind a disease-causing protein selected from: amyloid beta,oligomeric amyloid beta, amyloid beta plaques, amyloid precursor proteinor fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein,C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prionprotein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin,ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, Lewy body,atrial natriuretic factor, islet amyloid polypeptide, insulin,apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin,lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin,immunoglobulin light chain AL, S-IBM protein, Repeat-associated non-ATG(RAN) translation products, DiPeptide repeat (DPR) peptides,glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeatpeptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA)repeat peptides, ubiquitin, and proline-arginine (PR) repeat peptides,and any combination thereof; or with one or more antibodies that bind animmunomodulatory protein selected from: PD1/PDL1, CD40, OX40, ICOS,CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4,HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4,A2AR, LAG3, DR-5, CD2, CD5, CD39, CD73, phosphatidylserine, and anycombination thereof.

In some embodiments, antibody fragments of the present disclosure may befunctional fragments that bind the same epitope as any of theanti-Siglec-9 antibodies of the present disclosure. In some embodiments,the antibody fragments are miniaturized versions of the anti-Siglec-9antibodies or antibody fragments of the present disclosure that have thesame epitope of the corresponding full-length antibody, but have muchsmaller molecule weight. Such miniaturized anti-Siglec-9 antibodyfragments may have better brain penetration ability and a shorterhalf-life, which is advantageous for imaging and diagnostic utilities(see e.g., Lütje S et al., Bioconjug Chem. 2014 Feb. 19; 25(2):335-41;Tavaré R et al., Proc Natl Acad Sci US A. 2014 Jan. 21; 111(3):1108-13;and Wiehr S et al., Prostate. 2014 May; 74(7):743-55). Accordingly, insome embodiments, anti-Siglec-9 antibody fragments of the presentdisclosure have better brain penetration as compared to theircorresponding full-length antibodies and/or have a shorter half-life ascompared to their corresponding full-length antibodies.

Antibody Frameworks

Any of the antibodies described herein further include a framework. Insome embodiments, the framework is a human immunoglobulin framework. Forexample, in some embodiments, an antibody (e.g., an anti-Siglec-9antibody) comprises HVRs as in any of the above embodiments and furthercomprises an acceptor human framework, e.g., a human immunoglobulinframework or a human consensus framework. Human immunoglobulinframeworks may be part of the human antibody, or a non-human antibodymay be humanized by replacing one or more endogenous frameworks withhuman framework region(s). Human framework regions that may be used forhumanization include but are not limited to: framework regions selectedusing the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296(1993)); framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285(1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature(somatically mutated) framework regions or human germline frameworkregions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633(2008)); and framework regions derived from screening FR libraries (see,e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok etal., J. Biol. Chem. 271:22611-22618 (1996)).

In some embodiments, an antibody comprises a light chain variable regioncomprising an HVR-L1, an HVR-L2, and an HVR-L3 of the present disclosureand one, two, three or four of the light chain framework regions asshown in Table 4A. In some embodiments, an antibody comprises a heavychain variable region comprising an HVR-H1, an HVR-H2, and an HVR-H3 ofthe present disclosure and one, two, three or four of the heavy chainframework regions as shown in Table 3B. In some embodiments, an antibodycomprises a light chain variable region comprising an HVR-L1, an HVR-L2,and an HVR-L3 of the present disclosure and one, two, three or four ofthe light chain framework regions as shown in Table 4A and furthercomprises a heavy chain variable region comprising an HVR-H1, an HVR-H2,and an HVR-H3 of the present disclosure and one, two, three or four ofthe heavy chain framework regions as shown in Table 4B.

Antibody Preparation

Anti-Siglec-9 antibodies of the present disclosure can encompasspolyclonal antibodies, monoclonal antibodies, humanized and chimericantibodies, human antibodies, antibody fragments (e.g., Fab, Fab′-SH,Fv, scFv, and F(ab′)₂), bispecific and polyspecific antibodies,multivalent antibodies, heteroconjugate antibodies, conjugatedantibodies, library derived antibodies, antibodies having modifiedeffector functions, fusion proteins containing an antibody portion, andany other modified configuration of the immunoglobulin molecule thatincludes an antigen recognition site, such as an epitope having aminoacid residues of a Siglec-9 protein of the present disclosure, includingglycosylation variants of antibodies, amino acid sequence variants ofantibodies, and covalently modified antibodies. The anti-Siglec-9antibodies may be human, murine, rat, or of any other origin (includingchimeric or humanized antibodies).

(1) Polyclonal Antibodies

Polyclonal antibodies, such as polyclonal anti-Siglec-9 antibodies, aregenerally raised in animals by multiple subcutaneous (sc) orintraperitoneal (ip) injections of the relevant antigen and an adjuvant.It may be useful to conjugate the relevant antigen (e.g., a purified orrecombinant Siglec-9 protein of the present disclosure) to a proteinthat is immunogenic in the species to be immunized, e.g., keyhole limpethemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybeantrypsin inhibitor, using a bifunctional or derivatizing agent, e.g.,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are independently lower alkyl groups. Examples of adjuvants which may beemployed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). Theimmunization protocol may be selected by one skilled in the art withoutundue experimentation.

The animals are immunized against the desired antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg (for rabbits) or 5μg (for mice) of the protein or conjugate with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to fourteen days later,the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Conjugates also can bemade in recombinant cell culture as protein fusions. Also, aggregatingagents such as alum are suitable to enhance the immune response.

(2) Monoclonal Antibodies

Monoclonal antibodies, such as monoclonal anti-Siglec-9 antibodies, areobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translational modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies.

For example, the monoclonal anti-Siglec-9 antibodies may be made usingthe hybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization (e.g., apurified or recombinant Siglec-9 protein of the present disclosure).Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The immunizing agent will typically include the antigenic protein (e.g.,a purified or recombinant Siglec-9 protein of the present disclosure) ora fusion variant thereof. Generally peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, while spleen orlymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress (1986), pp. 59-103.

Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine or human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells thusprepared are seeded and grown in a suitable culture medium thatpreferably contains one or more substances that inhibit the growth orsurvival of the unfused, parental myeloma cells. For example, if theparental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which are substances that prevent the growth ofHGPRT-deficient cells.

Preferred immortalized myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors (available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA), as well asSP-2 cells and derivatives thereof (e.g., X63-Ag8-653) (available fromthe American Type Culture Collection, Manassas, Va. USA). Human myelomaand mouse-human heteromyeloma cell lines have also been described forthe production of human monoclonal antibodies (Kozbor, J. Immunol.,133:3001 (1984); Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen (e.g.,a Siglec-9 protein of the present disclosure). Preferably, the bindingspecificity of monoclonal antibodies produced by hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The culture medium in which the hybridoma cells are cultured can beassayed for the presence of monoclonal antibodies directed against thedesired antigen (e.g., a Siglec-9 protein of the present disclosure).Preferably, the binding affinity and specificity of the monoclonalantibody can be determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linked assay(ELISA). Such techniques and assays are known in the in art. Forexample, binding affinity may be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as tumors in a mammal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose chromatography, hydroxylapatitechromatography, gel electrophoresis, dialysis, affinity chromatography,and other methods as described above.

Anti-Siglec-9 monoclonal antibodies may also be made by recombinant DNAmethods, such as those disclosed in U.S. Pat. No. 4,816,567, and asdescribed above. DNA encoding the monoclonal antibodies is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that specifically bind to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,in order to synthesize monoclonal antibodies in such recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opin. Immunol.,5:256-262 (1993) and Plückthun, Immunol. Rev. 130:151-188 (1992).

In certain embodiments, anti-Siglec-9 antibodies can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)described the isolation of murine and human antibodies, respectively,from phage libraries. Subsequent publications describe the production ofhigh affinity (nanomolar (“nM”) range) human antibodies by chainshuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nucl. AcidsRes., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies of desired specificity (e.g., thosethat bind a Siglec-9 protein of the present disclosure).

The DNA encoding antibodies or fragments thereof may also be modified,for example, by substituting the coding sequence for human heavy- andlight-chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA,81:6851 (1984)), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Typically such non-immunoglobulin polypeptides aresubstituted for the constant domains of an antibody, or they aresubstituted for the variable domains of one antigen-combining site of anantibody to create a chimeric bivalent antibody comprising oneantigen-combining site having specificity for an antigen and anotherantigen-combining site having specificity for a different antigen.

The monoclonal antibodies described herein (e.g., anti-Siglec-9antibodies of the present disclosure or fragments thereof) may bymonovalent, the preparation of which is well known in the art. Forexample, one method involves recombinant expression of immunoglobulinlight chain and a modified heavy chain. The heavy chain is truncatedgenerally at any point in the Fc region so as to prevent heavy chaincrosslinking. Alternatively, the relevant cysteine residues may besubstituted with another amino acid residue or are deleted so as toprevent crosslinking. In vitro methods are also suitable for preparingmonovalent antibodies. Digestion of antibodies to produce fragmentsthereof, particularly Fab fragments, can be accomplished using routinetechniques known in the art.

Chimeric or hybrid anti-Siglec-9 antibodies also may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide-exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

(3) Humanized Antibodies

Anti-Siglec-9 antibodies of the present disclosure or antibody fragmentsthereof may further include humanized or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such asFab, Fab′-SH, Fv, scFv, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementaritydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit having the desired specificity, affinity and capacity. In someinstances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Humanized antibodies mayalso comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally willalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. Jones et al., Nature321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988) andPresta, Curr. Opin. Struct. Biol. 2: 593-596 (1992).

Methods for humanizing non-human anti-Siglec-9 antibodies are well knownin the art. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers, Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536(1988), or through substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody. Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. Carter et al., Proc. Nat'l Acad. Sci.USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993).

Furthermore, it is important that antibodies be humanized with retentionof high affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analyzing the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen or antigens (e.g., Siglec-9 proteins of the presentdisclosure), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding.

Various forms of the humanized anti-Siglec-9 antibody are contemplated.For example, the humanized anti-Siglec-9 antibody may be an antibodyfragment, such as an Fab, which is optionally conjugated with one ormore cytotoxic agent(s) in order to generate an immunoconjugate.Alternatively, the humanized anti-Siglec-9 antibody may be an intactantibody, such as an intact IgG1 antibody.

(4) Human Antibodies

Alternatively, human anti-Siglec-9 antibodies can be generated. Forexample, it is now possible to produce transgenic animals (e.g., mice)that are capable, upon immunization, of producing a full repertoire ofhuman antibodies in the absence of endogenous immunoglobulin production.The homozygous deletion of the antibody heavy-chain joining region(J_(H)) gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Nat'l Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immunol., 7:33 (1993); U.S. Pat. No. 5,591,669 and WO97/17852.

Alternatively, phage display technology can be used to produce humananti-Siglec-9 antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires from unimmunizeddonors. McCafferty et al., Nature 348:552-553 (1990); Hoogenboom andWinter, J. Mol. Biol. 227: 381 (1991). According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell,David J., Curr. Opin Struct. Biol. 3:564-571 (1993). Several sources ofV-gene segments can be used for phage display. Clackson et al., Nature352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodiesfrom a small random combinatorial library of V genes derived from thespleens of immunized mice. A repertoire of V genes from unimmunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Marks et al., J. Mol. Biol. 222:581-597(1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S.Pat. Nos. 5,565,332 and 5,573,905. Additionally, yeast displaytechnology can be used to produce human anti-Siglec-9 antibodies andantibody fragments in vitro (e.g., WO 2009/036379; WO 2010/105256; WO2012/009568; US 2009/0181855; US 2010/0056386; and Feldhaus and Siegel(2004) J. Immunological Methods 290:69-80). In other embodiments,ribosome display technology can be used to produce human anti-Siglec-9antibodies and antibody fragments in vitro (e.g., Roberts and Szostak(1997) Proc Natl Acad Sci 94:12297-12302; Schaffitzel et al. (1999) J.Immunolical Methods 231:119-135; Lipovsek and Plückthun (2004) J.Immunological Methods 290:51-67).

The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human anti-Siglec-9 monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol. 147(1): 86-95 (1991). Similarly,human anti-Siglec-9 antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806,5,569,825, 5,625,126, 5,633,425, 5,661,016 and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-13 (1994), Fishwild et al., Nature Biotechnology 14: 845-51(1996), Neuberger, Nature Biotechnology 14: 826 (1996) and Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

Finally, human anti-Siglec-9 antibodies may also be generated in vitroby activated B-cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

(5) Antibody Fragments

In certain embodiments there are advantages to using anti-Siglec-9antibody fragments, rather than whole anti-Siglec-9 antibodies. Smallerfragment sizes allow for rapid clearance and better brain penetration.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Method. 24:107-117 (1992); and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host cells, for example, using nucleic acids encodinganti-Siglec-9 antibodies of the present disclosure. Fab, Fv and scFvantibody fragments can all be expressed in and secreted from E. coli,thus allowing the straightforward production of large amounts of thesefragments. A anti-Siglec-9 antibody fragments can also be isolated fromthe antibody phage libraries as discussed above. Alternatively, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)2 fragments (Carter et al., Bio/Technology 10:163-167(1992)). According to another approach, F(ab′)₂ fragments can beisolated directly from recombinant host cell culture. Production of Faband F(ab′)₂ antibody fragments with increased in vivo half-lives aredescribed in U.S. Pat. No. 5,869,046. In other embodiments, the antibodyof choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S.Pat. Nos. 5,571,894 and 5,587,458. The anti-Siglec-9 antibody fragmentmay also be a “linear antibody,” e.g., as described in U.S. Pat. No.5,641,870. Such linear antibody fragments may be monospecific orbispecific.

(6) Bispecific and Polyspecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes, including those onthe same or another protein (e.g., one or more Siglec-9 proteins of thepresent disclosure). Alternatively, one part of a BsAb can be armed tobind to the target Siglec-9 antigen, and another can be combined with anarm that binds to a second protein. Such antibodies can be derived fromfull length antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy-chain/light chain pairs,where the two chains have different specificities. Millstein et al.,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. It is preferred tohave the first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyhalf of the bispecific molecules provides for an easy way of separation.This approach is disclosed in WO 94/04690. For further details ofgenerating bispecific antibodies, see, for example, Suresh et al.,Methods in Enzymology 121: 210 (1986).

According to another approach described in WO 96/27011 or U.S. Pat. No.5,731,168, the interface between a pair of antibody molecules can beengineered to maximize the percentage of heterodimers which arerecovered from recombinant cell culture. The preferred interfacecomprises at least a part of the C_(H)3 region of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g., tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chains(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g., alanine or threonine). Thisprovides a mechanism for increasing the yield of the heterodimer overother unwanted end-products such as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-TNB derivative to form the bispecificantibody. The bispecific antibodies produced can be used as agents forthe selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describes the production of fully humanized bispecificantibody F(ab′)₂ molecules. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Nat'l Acad. Sci. USA, 90: 6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific/bivalentantibody fragments by the use of single-chain Fv (sFv) dimers has alsobeen reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven molecule (e.g., a Siglec-9 protein of the present disclosure).Alternatively, an arm targeting a Siglec-9 signaling component may becombined with an arm which binds to a triggering molecule on a leukocytesuch as a T cell receptor molecule (e.g., CD2, CD3, CD28 or B7), or Fcreceptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) andFcγRIII (CD16) so as to focus cellular defense mechanisms to the cellexpressing the particular protein. Bispecific antibodies may also beused to localize cytotoxic agents to cells which express a particularprotein. Such antibodies possess a protein-binding arm and an arm whichbinds a cytotoxic agent or a radionuclide chelator, such as EOTUBE,DPTA, DOTA or TETA. Another bispecific antibody of interest binds theprotein of interest and further binds tissue factor (TF).

(7) Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The anti-Siglec-9 antibodies of the present disclosureor antibody fragments thereof can be multivalent antibodies (which areother than of the IgM class) with three or more antigen binding sites(e.g., tetravalent antibodies), which can be readily produced byrecombinant expression of nucleic acid encoding the polypeptide chainsof the antibody. The multivalent antibody can comprise a dimerizationdomain and three or more antigen binding sites. The preferreddimerization domain comprises an Fc region or a hinge region. In thisscenario, the antibody will comprise an Fc region and three or moreantigen binding sites amino-terminal to the Fc region. The preferredmultivalent antibody herein contains three to about eight, butpreferably four, antigen binding sites. The multivalent antibodycontains at least one polypeptide chain (and preferably two polypeptidechains), wherein the polypeptide chain or chains comprise two or morevariable domains. For instance, the polypeptide chain or chains maycomprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. Similarly, the polypeptide chain or chains may compriseV_(H)-C_(H)1-flexible linker-V_(H)-C_(H)1-Fc region chain; orV_(H)—C_(H)1-V_(H)-C_(H)1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain. The multivalentantibodies may recognize the Siglec-9 antigen as well as, withoutlimitation, additional antigens A beta peptide, antigen or an alphasynuclain protein antigen or, Tau protein antigen or, TDP-43 proteinantigen or, prion protein antigen or, huntingtin protein antigen, orRAN, translation Products antigen, including the DiPeptide Repeats,(DPRs peptides) composed of glycine-alanine (GA), glycine-proline (GP),glycine-arginine (GR), proline-alanine (PA), or proline-arginine (PR),insulin receptor, insulin like growth factor receptor, transferrinreceptor, or any other antigen that facilitates antibody transfer acrossthe blood brain barrier.

(8) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentdisclosure. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies (e.g., anti-Siglec-9 antibodies of the presentdisclosure or antibody fragments thereof). For example, one of theantibodies in the heteroconjugate can be coupled to avidin, the other tobiotin. Such antibodies have, for example, been proposed to targetimmune system cells to unwanted cells, U.S. Pat. No. 4,676,980, and havebeen used to treat HIV infection. International Publication Nos. WO91/00360, WO 92/200373 and EP 0308936. It is contemplated that theantibodies may be prepared in vitro using known methods in syntheticprotein chemistry, including those involving crosslinking agents. Forexample, immunotoxins may be constructed using a disulfide exchangereaction or by forming a thioether bond. Examples of suitable reagentsfor this purpose include iminothiolate and methyl-4-mercaptobutyrimidateand those disclosed, for example, in U.S. Pat. No. 4,676,980.Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

(9) Effector Function Engineering

It may also be desirable to modify an anti-Siglec-9 antibody of thepresent disclosure to modify effector function and/or to increase serumhalf-life of the antibody. For example, the Fc receptor binding site onthe constant region may be modified or mutated to remove or reducebinding affinity to certain Fc receptors, such as FcγRI, FcγRII, and/orFcγRIII. In some embodiments, the effector function is impaired byremoving N-glycosylation of the Fc region (e.g., in the CH 2 domain ofIgG) of the antibody. In some embodiments, the effector function isimpaired by modifying regions such as 233-236, 297, and/or 327-331 ofhuman IgG as described in PCT WO 99/58572 and Armour et al., MolecularImmunology 40: 585-593 (2003); Reddy et al., J. Immunology 164:1925-1933(2000).

To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

(10) Other Amino Acid Sequence Modifications

Amino acid sequence modifications of anti-Siglec-9 antibodies of thepresent disclosure, or antibody fragments thereof, are alsocontemplated. For example, it may be desirable to improve the bindingaffinity and/or other biological properties of the antibodies orantibody fragments. Amino acid sequence variants of the antibodies orantibody fragments are prepared by introducing appropriate nucleotidechanges into the nucleic acid encoding the antibodies or antibodyfragments, or by peptide synthesis. Such modifications include, forexample, deletions from, and/or insertions into and/or substitutions of,residues within the amino acid sequences of the antibody. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics (i.e., the ability to bind or physicallyinteract with a Siglec-9 protein of the present disclosure). The aminoacid changes also may alter post-translational processes of theantibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theanti-Siglec-9 antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells in Science, 244:1081-1085 (1989). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (most preferably alanine or polyalanine) to affect the interactionof the amino acids with the target antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, alanine scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- (“N”) and/or carboxy-(“C”) terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include an antibody with an N-terminalmethionyl residue or the antibody fused to a cytotoxic polypeptide.Other insertional variants of the antibody molecule include the fusionto the N- or C-terminus of the antibody to an enzyme or a polypeptidewhich increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin the Table E below under the heading of “preferred substitutions”. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table E,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE E Amino acid substitutions Preferred Original Residue ExemplarySubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala alaHis (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K)arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) Ser ser Trp (W) tyr; phetyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala;norleucine leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment, such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human anti-Siglec-9 antibody). Generally,the resulting variant(s) selected for further development will haveimproved biological properties relative to the parent antibody fromwhich they are generated. A convenient way for generating suchsubstitutional variants involves affinity maturation using phagedisplay. Briefly, several hypervariable region sites (e.g., 6-7 sites)are mutated to generate all possible amino substitutions at each site.The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g., bindingaffinity) as herein disclosed. In order to identify candidatehypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the antibodyand the antigen (e.g., a Siglec-9 protein of the present disclosure).Such contact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and antibodies with superior properties in one ormore relevant assays may be selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-IgE antibody are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the antibodies (e.g.,anti-Siglec-9 antibodies of the present disclosure) or antibodyfragments.

(11) Antibody Conjugates

Anti-Siglec-9 antibodies of the present disclosure, or antibodyfragments thereof, can be conjugated to a detectable marker, a toxin, ora therapeutic agent. Any suitable method known in the art forconjugating molecules, such as a detectable marker, a toxin, or atherapeutic agent to antibodies may be used.

For example, drug conjugation involves coupling of a biological activecytotoxic (anticancer) payload or drug to an antibody that specificallytargets a certain tumor marker (e.g. a protein that, ideally, is only tobe found in or on tumor cells). Antibodies track these proteins down inthe body and attach themselves to the surface of cancer cells. Thebiochemical reaction between the antibody and the target protein(antigen) triggers a signal in the tumor cell, which then absorbs orinternalizes the antibody together with the cytotoxin. After the ADC isinternalized, the cytotoxic drug is released and kills the cancer. Dueto this targeting, ideally the drug has lower side effects and gives awider therapeutic window than other chemotherapeutic agents. Technics toconjugate antibodies are disclosed are known in the art (see, e.g., Janede Lartigue, OncLive Jul. 5, 2012; ADC Review on antibody-drugconjugates; and Ducry et al., (2010). Bioconjugate Chemistry 21 (1):5-13).

In some embodiments, an anti-Siglec-9 antibody of the present disclosuremay be conjugated to a toxin selected from ricin, ricin A-chain,doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin,curicin, crotin, calicheamicin, Saponaria officinalis inhibitor,glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin,duocarmycins, dolastatin, cc1065, and a cisplatin.

(12) Other Antibody Modifications

Anti-Siglec-9 antibodies of the present disclosure, or antibodyfragments thereof, can be further modified to contain additionalnon-proteinaceous moieties that are known in the art and readilyavailable. Preferably, the moieties suitable for derivatization of theantibody are water-soluble polymers. Non-limiting examples ofwater-soluble polymers include, but are not limited to, polyethyleneglycol (PEG), copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, polypropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer is attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc. Such techniques and othersuitable formulations are disclosed in Remington: The Science andPractice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., PhiladelphiaCollege of Pharmacy and Science (2000).

Binding Assays and Other Assays

Anti-Siglec-9 antibodies of the present disclosure may be tested forantigen binding activity, e.g., by known methods such as ELISA, surfaceplasmon resonance (SPR), Western blot, etc.

In some embodiments, competition assays may be used to identify anantibody that competes with any of the antibodies listed in Tables 2, 3,4A, 4B, 7A, and 7B, or selected from 2D4, 2D5, 5B1, 6B2, 6D8, 7H12, 5C6,12B12, and 17C2. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by any of the antibodies listed in Tables 2, 3, 4A, 4B, 7A, and7B, or selected from 2D4, 2D5, 5B 1, 6B2, 6D8, 7H12, 5C6, 12B12, and17C2. Detailed exemplary methods for mapping an epitope to which anantibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.).

In an exemplary competition assay, immobilized Siglec-9 or cellsexpressing Siglec-9 on a cell surface are incubated in a solutioncomprising a first labeled antibody that binds to Siglec-9 (e.g., humanor non-human primate) and a second unlabeled antibody that is beingtested for its ability to compete with the first antibody for binding toSiglec-9. The second antibody may be present in a hybridoma supernatant.As a control, immobilized Siglec-9 or cells expressing Siglec-9 isincubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to Siglec-9, excess unboundantibody is removed, and the amount of label associated with immobilizedSiglec-9 or cells expressing Siglec-9 is measured. If the amount oflabel associated with immobilized Siglec-9 or cells expressing Siglec-9is substantially reduced in the test sample relative to the controlsample, then that indicates that the second antibody is competing withthe first antibody for binding to Siglec-9. See, Harlow and Lane (1988)Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

Nucleic Acids, Vectors, and Host Cells

Anti-Siglec-9 antibodies of the present disclosure may be produced usingrecombinant methods and compositions, e.g., as described in U.S. Pat.No. 4,816,567. In some embodiments, isolated nucleic acids having anucleotide sequence encoding any of the anti-Siglec-9 antibodies of thepresent disclosure are provided. Such nucleic acids may encode an aminoacid sequence containing the VL and/or an amino acid sequence containingthe VH of the anti-Siglec-9 antibody (e.g., the light and/or heavychains of the antibody). In some embodiments, one or more vectors (e.g.,expression vectors) containing such nucleic acids are provided. In someembodiments, a host cell containing such nucleic acid is also provided.In some embodiments, the host cell contains (e.g., has been transducedwith): (1) a vector containing a nucleic acid that encodes an amino acidsequence containing the VL of the antibody and an amino acid sequencecontaining the VH of the antibody, or (2) a first vector containing anucleic acid that encodes an amino acid sequence containing the VL ofthe antibody and a second vector containing a nucleic acid that encodesan amino acid sequence containing the VH of the antibody. In someembodiments, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary(CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

Methods of making an anti-Siglec-9 antibody of the present disclosureare provided. In some embodiments, the method includes culturing a hostcell of the present disclosure containing a nucleic acid encoding theanti-Siglec-9 antibody, under conditions suitable for expression of theantibody. In some embodiments, the antibody is subsequently recoveredfrom the host cell (or host cell culture medium).

For recombinant production of an anti-Siglec-9 antibody of the presentdisclosure, a nucleic acid encoding the anti-Siglec-9 antibody isisolated and inserted into one or more vectors for further cloningand/or expression in a host cell. Such nucleic acid may be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody).

Suitable vectors containing a nucleic acid sequence encoding any of theanti-Siglec-9 antibodies of the present disclosure, or fragments thereofpolypeptides (including antibodies) described herein include, withoutlimitation, cloning vectors and expression vectors. Suitable cloningvectors can be constructed according to standard techniques, or may beselected from a large number of cloning vectors available in the art.While the cloning vector selected may vary according to the host cellintended to be used, useful cloning vectors generally have the abilityto self-replicate, may possess a single target for a particularrestriction endonuclease, and/or may carry genes for a marker that canbe used in selecting clones containing the vector. Suitable examplesinclude plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript(e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1,pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. Theseand many other cloning vectors are available from commercial vendorssuch as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a nucleic acid of the present disclosure. The expressionvector may replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, andexpression vector(s) disclosed in PCT Publication No. WO 87/04462.Vector components may generally include, but are not limited to, one ormore of the following: a signal sequence; an origin of replication; oneor more marker genes; suitable transcriptional controlling elements(such as promoters, enhancers and terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons.

The vectors containing the nucleic acids of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell. In some embodiments, the vector contains a nucleic acidcontaining one or more amino acid sequences encoding an anti-Siglec-9antibody of the present disclosure.

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells. For example,anti-Siglec-9 antibodies of the present disclosure may be produced inbacteria, in particular when glycosylation and Fe effector function arenot needed. For expression of antibody fragments and polypeptides inbacteria (e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; andCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli.). After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microorganisms, such asfilamentous fungi or yeast, are also suitable cloning or expressionhosts for antibody-encoding vectors, including fungi and yeast strainswhose glycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li etal., Nat. Biotech. 24:210-215 (2006)).

Suitable host cells for the expression of glycosylated antibody can alsobe derived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts (e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,and 6,417,429, describing PLANTIBODIES™ technology for producingantibodies in transgenic plants.).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Siglec-9 Activities

PI3K Activation

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may induce PI3Kactivation after binding to a Siglec-9 protein expressed in a cell.

PI3Ks are a family of related intracellular signal transducer kinasescapable of phosphorylating the 3-position hydroxyl group of the inositolring of phosphatidylinositol (PtdIns). The PI3K family is divided intothree different classes (Class I, Class II, and Class III) based onprimary structure, regulation, and in vitro lipid substrate specificity.

Activated PI3K produces various 3-phosphorylated phosphoinositides,including without limitation, PtdIns3P, PtdIns(3,4)P2, PtdIns(3,5)P2,and PtdIns(3,4,5)P3. These 3-phosphorylated phosphoinositides functionin a mechanism by which signaling proteins are recruited to variouscellular membranes. These signaling proteins containphosphoinositide-binding domains, including without limitation, PXdomains, pleckstrin homology domains (PH domains), and FYVE domains. Anymethod known in the art for determining PI3K activation may be used.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with decreased levels of PI3K activity, including, withoutlimitation, dementia, frontotemporal dementia, Alzheimer's disease,vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normalpressure hydrocephalus, amyotrophic lateral sclerosis, Huntington'sdisease, taupathy disease, Nasu-Hakola disease, stroke, acute trauma,chronic trauma, lupus, acute and chronic colitis, rheumatoid arthritis,wound healing, Crohn's disease, inflammatory bowel disease, ulcerativecolitis, obesity, malaria, essential tremor, central nervous systemlupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies,multiple system atrophy, Shy-Drager syndrome, progressive supranuclearpalsy, cortical basal ganglionic degeneration, acute disseminatedencephalomyelitis, granulomartous disorders, sarcoidosis, diseases ofaging, seizures, spinal cord injury, traumatic brain injury, age relatedmacular degeneration, glaucoma, retinitis pigmentosa, retinaldegeneration, respiratory tract infection, sepsis, eye infection,systemic infection, lupus, arthritis, multiple sclerosis, low bonedensity, osteoporosis, osteogenesis, osteopetrotic disease, Paget'sdisease of bone, and cancer including bladder cancer, brain cancer,breast cancer, colon cancer, rectal cancer, endometrial cancer, kidneycancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer,melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer,ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyeloid leukemia (CML), multiple myeloma, polycythemia vera, essentialthrombocytosis, primary or idiopathic myelofibrosis, primary oridiopathic myelosclerosis, myeloid-derived tumors, tumors that expressSiglec-9, thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

Modulated Expression of Cytokines

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may modulate (e.g.,increase or decrease) pro-inflammatory mediators in the brain afterbinding to a Siglec-9 protein expressed on a cell surface. In certainembodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, modulate theexpression of cytokines (e.g., proinflammatory mediators) and/or reducethe expression of anti-inflammatory mediators after binding to aSiglec-9 protein expressed in a cell.

Inflammation is part of a complex biological response of vasculartissues to harmful stimuli, such as pathogens, damaged cells, andirritants. The classical signs of acute inflammation are pain, heat,redness, and swelling. Inflammation is an immune response that protectsan organism by limiting the site of injury or clearing an infection byrecruiting and activating cells of the immune system. The inflammatoryresponse is tightly regulated and restricted in its duration andseverity to avoid causing damage to the organism. Inflammation can beclassified as either acute or chronic. Acute inflammation is driven bythe innate immune response, which initially recognizes harmful stimuliand recruits leukocytes from the blood into the injured tissues. Acascade of biochemical events, including cytokine and chemokine release,propagates the inflammatory response, involving the local vascularsystem, the immune system, and various cells within the injured tissue.Chronic inflammation is prolonged and persistent which leads to aprogressive shift in the type of immune cells participating in theinflammatory response. Chronic inflammation is characterized byprogressive destruction and fibrosis of the tissue as a result of theinflammatory process.

As used herein, anti-inflammatory mediators are proteins involved eitherdirectly or indirectly (e.g., by way of an anti-inflammatory signalingpathway) in a mechanism that reduces, inhibits, or inactivates aninflammatory response. Any method known in the art for identifying andcharacterizing anti-inflammatory mediators may be used. Examples ofanti-inflammatory mediators include, without limitation, cytokines, suchas IL-4, IL-10, IL-13, IL-35, IL-16, IFN-alpha, TGF-beta, IL-1Ra, G-CSF,and soluble receptors for TNF-alpha or IL-6. Examples ofpro-inflammatory mediators include, without limitation, cytokines, suchas FN-α4, IFN-3, IL-10, TNF-α, IL-6, IL-8, CRP, IL-20 family members,LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-17, IL-18, IL-23,CXCL10, IL-33, CRP, IL-33, MCP-1, and MIP-1-beta.

In some embodiments, the Siglec-9 agents of the present disclosure, suchas anti-Siglec-9 antibodies of the present disclosure, may modulate(e.g., increase or decrease) expression of cytokines, such as IL-1b,IL-8, and TNF-α. In certain embodiments, modulated expression of thecytokines occurs in macrophages, neutrophils, natural killer (NK) cells,dendritic cells, monocytes, osteoclasts, Langerhans cells of skin,Kupffer cells, T cells, and/or microglial cells. Modulated expressionmay include, without limitation, an increase in gene expression, anincrease in transcriptional expression, or an increase in proteinexpression. Any method known in the art for determining gene, transcript(e.g., mRNA), and/or protein expression may be used. For example,Northern blot analysis may be used to determine cytokine gene expressionlevels, RT-PCR may be used to determine the level of cytokinetranscription, and Western blot analysis may be used to determinecytokine protein levels.

As used herein, a cytokine may have modulated expression if itsexpression in one or more cells of a subject treated with an Siglec-9agents of the present disclosure, such as anti-Siglec-9 antibodies ofthe present disclosure, is modulated as compared to the expression ofthe same cytokine expressed in one or more cells of a correspondingsubject that is not treated with the Siglec-9 agent. In someembodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may modulatecytokine expression in one or more cells of a subject by at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 100%, at least 110%, at least 115%, atleast 120%, at least 125%, at least 130%, at least 135%, at least 140%,at least 145%, at least 150%, at least 160%, at least 170%, at least180%, at least 190%, or at least 200% for example, as compared tocytokine expression in one or more cells of a corresponding subject thatis not treated with the Siglec-9 agent. In other embodiments, Siglec-9agents of the present disclosure, such as anti-Siglec-9 antibodies ofthe present disclosure, modulate cytokine expression in one or morecells of a subject by at least 1.5 fold, at least 1.6 fold, at least 1.7fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, atleast 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, atleast 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold,at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0fold, at least 9.5 fold, or at least 10 fold, for example, as comparedto cytokine expression in one or more cells of a corresponding subjectthat is not treated with the Siglec-9 agent.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are useful forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with abnormal levels of one or more pro-inflammatorymediators, including without limitation, dementia, frontotemporaldementia, Alzheimer's disease, vascular dementia, mixed dementia,Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophiclateral sclerosis, Huntington's disease, taupathy disease, Nasu-Hakoladisease, stroke, acute trauma, chronic trauma, lupus, acute and chroniccolitis, rheumatoid arthritis, wound healing, Crohn's disease,inflammatory bowel disease, ulcerative colitis, obesity, malaria,essential tremor, central nervous system lupus, Behcet's disease,Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,Shy-Drager syndrome, progressive supranuclear palsy, cortical basalganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, and cancerincluding bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

Modulated Expression of Pro-Inflammatory Mediators

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may modulate (e.g.,increase or decrease) the expression of pro-inflammatory mediators afterbinding to a Siglec-9 protein expressed in a cell.

As used herein, pro-inflammatory mediators are proteins involved eitherdirectly or indirectly (e.g., by way of pro-inflammatory signalingpathways) in a mechanism that induces, activates, promotes, or otherwiseincreases an inflammatory response. Any method known in the art foridentifying and characterizing pro-inflammatory mediators may be used.

Examples of pro-inflammatory mediators include, without limitation,cytokines, such as type I and II interferons, IL-1β, TNF-α, IL-6, IL-8,IL-20 family members, IL-33, LIF, OSM, CNTF, GM-CSF, IL-11, IL-12,IL-17, IL-18, and CRP.

In some embodiments, the anti-Siglec-9 antibodies of the presentdisclosure may modulate functional expression and/or secretion ofpro-inflammatory mediators, such as type I and 11 interferons, FN-c4,IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ,OSM, CNTF, GM-CSF, IL-11, IL-12, IL-17, IL-18, IL-23, CXCL10, IL-33,CRP, IL-33, MCP-1, and MIP-1-beta. In certain embodiments, modulatedexpression of the pro-inflammatory mediators occurs in macrophages,neutrophils, NK cells, dendritic cells, monocytes, osteoclasts,Langerhans cells of skin, Kupffer cells, T cells, and/or microglialcells. Modulated expression may include, without limitation, a modulatedgene expression, modulated transcriptional expression, or modulatedprotein expression. Any method known in the art for determining gene,transcript (e.g., mRNA), and/or protein expression may be used. Forexample, Northern blot analysis may be used to determinepro-inflammatory mediator gene expression levels, RT-PCR may be used todetermine the level of pro-inflammatory mediator transcription, andWestern blot analysis may be used to determine pro-inflammatory mediatorprotein levels.

In certain embodiments, pro-inflammatory mediators include inflammatorycytokines. Accordingly, in certain embodiments, the Siglec-9 agents ofthe present disclosure, such as anti-Siglec-9 antibodies of the presentdisclosure, may modulate secretion of one or more inflammatorycytokines. Examples of inflammatory cytokines whose secretion may bemodulated by the anti-Siglec-9 antibodies of the present disclosureinclude, without limitation, such as type I and II interferons, FN-α4,IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ,OSM, CNTF, GM-CSF, IL-11, IL-12, IL-17, IL-18, IL-23, CXCL10, IL-33,CRP, IL-33, MCP-1, and MIP-1-beta.

In certain embodiments, pro-inflammatory mediators include inflammatoryreceptors, proteins of the complement cascade, and/or receptors that areexpressed on immune cells. Accordingly, in certain embodiments, theSiglec-9 agents of the present disclosure, such as anti-Siglec-9antibodies of the present disclosure, may modulate expression of one ormore inflammatory receptors, proteins of the complement cascade, and/orreceptors that are expressed on immune cells. Examples of inflammatoryreceptors, proteins of the complement cascade, and/or receptors that areexpressed on immune cells whose expression may be modulated by theSiglec-9 agents of the present disclosure, such as anti-Siglec-9antibodies of the present disclosure, include, without limitation, CD86,CD80, CD83, C1qa, C1qB, C1qC, C1s, C1R, C4, C2, C3, ITGB2, HMOX1, LAT2,CASP1, CSTA, VSIG4, MS4A4A, C3AR1, GPX1, TyroBP, ALOX5AP, ITGAM, SLC7A7,CD4, ITGAX, and PYCARD.

As used herein, a pro-inflammatory mediator may have modulatedexpression if its expression in one or more cells of a subject treatedwith a Siglec-9 agent, such as an agonist anti-Siglec-9 antibody of thepresent disclosure is modulated (e.g., increased or decreased) ascompared to the expression of the same pro-inflammatory mediatorexpressed in one or more cells of a corresponding subject that is nottreated with the agonist anti-Siglec-9 antibody. In some embodiments,the anti-Siglec-9 antibody of the present disclosure may modulatepro-inflammatory mediator expression in one or more cells of a subjectby at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 100%, at least 110%, atleast 115%, at least 120%, at least 125%, at least 130%, at least 135%,at least 140%, at least 145%, at least 150%, at least 160%, at least170%, at least 180%, at least 190%, or at least 200% for example, ascompared to pro-inflammatory mediator expression in one or more cells ofa corresponding subject that is not treated with the anti-Siglec-9antibody. In other embodiments, the anti-Siglec-9 antibody may modulatepro-inflammatory mediator expression in one or more cells of a subjectby at least at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, atleast 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold,at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, atleast 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, atleast 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold,at least 9.5 fold, or at least 10 fold, for example, as compared topro-inflammatory mediator expression in one or more cells of acorresponding subject that is not treated with the anti-Siglec-9antibody.

In some embodiments, some Siglec-9 agents of the present disclosure,such as anti-Siglec-9 antibodies of the present disclosure, may beuseful for preventing, lowering the risk of, or treating conditionsand/or diseases associated with abnormal levels of one or morepro-inflammatory mediators, including without limitation, dementia,frontotemporal dementia, Alzheimer's disease, vascular dementia, mixeddementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus,amyotrophic lateral sclerosis, Huntington's disease, taupathy disease,Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acuteand chronic colitis, rheumatoid arthritis, wound healing, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, obesity,malaria, essential tremor, central nervous system lupus, Behcet'sdisease, Parkinson's disease, dementia with Lewy bodies, multiple systematrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticalbasal ganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, and cancerincluding bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

ERK Phosphorylation

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may induceextracellular signal-regulated kinase (ERK) phosphorylation afterbinding to a Siglec-9 protein expressed in a cell.

Extracellular-signal-regulated kinases (ERKs) are widely expressedprotein kinase intracellular signaling kinases that are involved in, forexample, the regulation of meiosis, mitosis, and postmitotic functionsin differentiated cells. Various stimuli, such as growth factors,cytokines, virus infection, ligands for heterotrimeric G protein-coupledreceptors, transforming agents, and carcinogens, activate ERK pathways.Phosphorylation of ERKs leads to the activation of their kinaseactivity.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with decreased levels of ERK phosphorylation, includingwithout limitation, dementia, frontotemporal dementia, Alzheimer'sdisease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease,normal pressure hydrocephalus, amyotrophic lateral sclerosis,Huntington's disease, taupathy disease, Nasu-Hakola disease, stroke,acute trauma, chronic trauma, lupus, acute and chronic colitis,rheumatoid arthritis, wound healing, Crohn's disease, inflammatory boweldisease, ulcerative colitis, obesity, malaria, essential tremor, centralnervous system lupus, Behcet's disease, Parkinson's disease, dementiawith Lewy bodies, multiple system atrophy, Shy-Drager syndrome,progressive supranuclear palsy, cortical basal ganglionic degeneration,acute disseminated encephalomyelitis, granulomartous disorders,sarcoidosis, diseases of aging, seizures, spinal cord injury, traumaticbrain injury, age related macular degeneration, glaucoma, retinitispigmentosa, retinal degeneration, respiratory tract infection, sepsis,eye infection, systemic infection, lupus, arthritis, multiple sclerosis,low bone density, osteoporosis, osteogenesis, osteopetrotic disease,Paget's disease of bone, and cancer including bladder cancer, braincancer, breast cancer, colon cancer, rectal cancer, endometrial cancer,kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lungcancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostatecancer, ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),chronic myeloid leukemia (CML), multiple myeloma, polycythemia vera,essential thrombocytosis, primary or idiopathic myelofibrosis, primaryor idiopathic myelosclerosis, myeloid-derived tumors, tumors thatexpress Siglec-9, thyroid cancer, infections, CNS herpes, parasiticinfections, Trypanosome infection, Cruzi infection, Pseudomonasaeruginosa infection, Leishmania donovani infection, group BStreptococcus infection, Campylobacter jejuni infection, Neisseriameningiditis infection, type I HIV, and Haemophilus influenza.

Syk Phosphorylation

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may induce spleentyrosine kinase (Syk) phosphorylation after binding to a Siglec-9protein expressed in a cell.

Spleen tyrosine kinase (Syk) is an intracellular signaling molecule thatfunctions downstream of Siglec-9 by phosphorylating several substrates,thereby facilitating the formation of a signaling complex leading tocellular activation and inflammatory processes.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with decreased levels of Syk phosphorylation, includingwithout limitation, dementia, frontotemporal dementia, Alzheimer'sdisease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease,normal pressure hydrocephalus, amyotrophic lateral sclerosis,Huntington's disease, taupathy disease, Nasu-Hakola disease, stroke,acute trauma, chronic trauma, lupus, acute and chronic colitis,rheumatoid arthritis, wound healing, Crohn's disease, inflammatory boweldisease, ulcerative colitis, obesity, malaria, essential tremor, centralnervous system lupus, Behcet's disease, Parkinson's disease, dementiawith Lewy bodies, multiple system atrophy, Shy-Drager syndrome,progressive supranuclear palsy, cortical basal ganglionic degeneration,acute disseminated encephalomyelitis, granulomartous disorders,sarcoidosis, diseases of aging, seizures, spinal cord injury, traumaticbrain injury, age related macular degeneration, glaucoma, retinitispigmentosa, retinal degeneration, respiratory tract infection, sepsis,eye infection, systemic infection, lupus, arthritis, multiple sclerosis,low bone density, osteoporosis, osteogenesis, osteopetrotic disease,Paget's disease of bone, and cancer including bladder cancer, braincancer, breast cancer, colon cancer, rectal cancer, endometrial cancer,kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lungcancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostatecancer, ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),chronic myeloid leukemia (CML), multiple myeloma, polycythemia vera,essential thrombocytosis, primary or idiopathic myelofibrosis, primaryor idiopathic myelosclerosis, myeloid-derived tumors, tumors thatexpress Siglec-9, thyroid cancer, infections, CNS herpes, parasiticinfections, Trypanosome infection, Cruzi infection, Pseudomonasaeruginosa infection, Leishmania donovani infection, group BStreptococcus infection, Campylobacter jejuni infection, Neisseriameningiditis infection, type I HIV, and Haemophilus influenza.

Siglec-9 Phosphorylation

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may transientlyinduce Siglec-9 phosphorylation of Tyr-433 and Tyr-456 by a by Srcfamily tyrosine kinase such as Src, Syk, Fyn, Fgr, Lck, Hck, Blk, Lyn,and Frk after binding to a Siglec-9 protein expressed in a cell.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with decreased levels of Siglec-9 phosphorylation, includingwithout limitation, dementia, frontotemporal dementia, Alzheimer'sdisease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease,normal pressure hydrocephalus, amyotrophic lateral sclerosis,Huntington's disease, taupathy disease, Nasu-Hakola disease, stroke,acute trauma, chronic trauma, lupus, acute and chronic colitis,rheumatoid arthritis, wound healing, Crohn's disease, inflammatory boweldisease, ulcerative colitis, obesity, malaria, essential tremor, centralnervous system lupus, Behcet's disease, Parkinson's disease, dementiawith Lewy bodies, multiple system atrophy, Shy-Drager syndrome,progressive supranuclear palsy, cortical basal ganglionic degeneration,acute disseminated encephalomyelitis, granulomartous disorders,sarcoidosis, diseases of aging, seizures, spinal cord injury, traumaticbrain injury, age related macular degeneration, glaucoma, retinitispigmentosa, retinal degeneration, respiratory tract infection, sepsis,eye infection, systemic infection, lupus, arthritis, multiple sclerosis,low bone density, osteoporosis, osteogenesis, osteopetrotic disease,Paget's disease of bone, and cancer including bladder cancer, braincancer, breast cancer, colon cancer, rectal cancer, endometrial cancer,kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lungcancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostatecancer, ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),chronic myeloid leukemia (CML), multiple myeloma, polycythemia vera,essential thrombocytosis, primary or idiopathic myelofibrosis, primaryor idiopathic myelosclerosis, myeloid-derived tumors, tumors thatexpress Siglec-9, thyroid cancer, infections, CNS herpes, parasiticinfections, Trypanosome infection, Cruzi infection, Pseudomonasaeruginosa infection, Leishmania donovani infection, group BStreptococcus infection, Campylobacter jejuni infection, Neisseriameningiditis infection, type I HIV, and Haemophilus influenza.

Phosphorylation of ITAM Motif Containing Receptors

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may inducephosphorylate ITAM motif-containing receptors, such as TREM1, TREM2,Sirp beta, FcgR, DAP10, and DAP12, after binding to a Siglec-9 proteinexpressed in a cell.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with decreased levels of phosphorylation of ITAMmotif-containing receptors, including without limitation, dementia,frontotemporal dementia, Alzheimer's disease, vascular dementia, mixeddementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus,amyotrophic lateral sclerosis, Huntington's disease, taupathy disease,Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acuteand chronic colitis, rheumatoid arthritis, wound healing, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, obesity,malaria, essential tremor, central nervous system lupus, Behcet'sdisease, Parkinson's disease, dementia with Lewy bodies, multiple systematrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticalbasal ganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, and cancerincluding bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

Modulated Expression of C—C Chemokine Receptor 7

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may modulateexpression of C—C chemokine receptor 7 (CCR7) after binding to aSiglec-9 protein expressed in a cell. Modulated (e.g., increased ordecreased) expression may include, without limitation, modulation ingene expression, modulation in transcriptional expression, or modulationin protein expression. Any method known in the art for determining gene,transcript (e.g., mRNA), and/or protein expression may be used. Forexample, Northern blot analysis may be used to determineanti-inflammatory mediator gene expression levels, RT-PCR may be used todetermine the level of anti-inflammatory mediator transcription, andWestern blot analysis may be used to determine anti-inflammatorymediator protein levels.

C—C chemokine receptor 7 (CCR7) is a member of the G protein-coupledreceptor family. CCR7 is expressed in various lymphoid tissues and canactivate B cells and T cells. In some embodiments, CCR7 may modulate themigration of memory T cells to secondary lymphoid organs, such as lymphnodes. In other embodiments, CCR7 may stimulate dendritic cellmaturation. CCR7 is a receptor protein that can hind the chemokine (C—Cmotif) ligands CCL19/ELC and CCL21.

As used herein, CCR7 may have modulated expression if its expression inone or more cells of a subject treated with an Siglec-9 agents of thepresent disclosure, such as anti-Siglec-9 antibodies of the presentdisclosure, is modulated (e.g., increased or decreased) as compared tothe expression of CCR7 expressed in one or more cells of a correspondingsubject that is not treated with the Siglec-9 agent. In someembodiments, an Siglec-9 agent of the present disclosure, such as ananti-Siglec-9 antibody of the present disclosure, may modulate CCR7expression in one or more cells of a subject by at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 100%, at least 110%, at least 115%, at least120%, at least 125%, at least 130%, at least 135%, at least 140%, atleast 145%, at least 150%, at least 160%, at least 170%, at least 180%,at least 190%, or at least 200% for example, as compared to CCR7expression in one or more cells of a corresponding subject that is nottreated with the Siglec-9 agent. In other embodiments, an Siglec-9 agentof the present disclosure, such as an anti-Siglec-9 antibody of thepresent disclosure, modulates CCR7 expression in one or more cells of asubject by at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, atleast 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold,at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, atleast 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, atleast 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold,at least 9.5 fold, or at least 10 fold, for example, as compared to CCR7expression in one or more cells of a corresponding subject that is nottreated with the Siglec-9 agent.

In some embodiments, increased expression of CCR7 occurs in macrophages,neutrophils, NK cells, dendritic cells, and/or microglial cells.Increased expression of CCR7 may induce microglial cell chemotaxistoward cells expressing the chemokines CCL19 and CCL21. Accordingly, incertain embodiments, anti-Siglec-9 antibodies of the present disclosuremay induce microglial cell chemotaxis toward CCL19 and CCL21 expressingcells.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are useful forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with abnormal levels of CCR7, including without limitation,dementia, frontotemporal dementia, Alzheimer's disease, vasculardementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressurehydrocephalus, amyotrophic lateral sclerosis, Huntington's disease,taupathy disease, Nasu-Hakola disease, stroke, acute trauma, chronictrauma, lupus, acute and chronic colitis, rheumatoid arthritis, woundhealing, Crohn's disease, inflammatory bowel disease, ulcerativecolitis, obesity, malaria, essential tremor, central nervous systemlupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies,multiple system atrophy, Shy-Drager syndrome, progressive supranuclearpalsy, cortical basal ganglionic degeneration, acute disseminatedencephalomyelitis, granulomartous disorders, sarcoidosis, diseases ofaging, seizures, spinal cord injury, traumatic brain injury, age relatedmacular degeneration, glaucoma, retinitis pigmentosa, retinaldegeneration, respiratory tract infection, sepsis, eye infection,systemic infection, lupus, arthritis, multiple sclerosis, low bonedensity, osteoporosis, osteogenesis, osteopetrotic disease, Paget'sdisease of bone, and cancer including bladder cancer, brain cancer,breast cancer, colon cancer, rectal cancer, endometrial cancer, kidneycancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer,melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer,ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyeloid leukemia (CML), multiple myeloma, polycythemia vera, essentialthrombocytosis, primary or idiopathic myelofibrosis, primary oridiopathic myelosclerosis, myeloid-derived tumors, tumors that expressSiglec-9, thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

Enhancement or Normalization of the Ability of Bone Marrow-DerivedDendritic Cells to Induce Antigen-Specific T Cell Proliferation

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may enhance and/ornormalize the ability of bone marrow-derived dendritic cells to induceantigen-specific T cell proliferation after binding to a Siglec-9protein expressed in a cell.

In some embodiments, Siglec-9 agents of the present disclosure, such asantagonist anti-Siglec-9 antibodies of the present disclosure, mayenhance and/or normalize the ability of bone marrow-derived dendriticcells to induce antigen-specific T cell proliferation in one or morebone marrow-derived dendritic cells of a subject by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 100%, at least 110%, at least 115%, atleast 120%, at least 125%, at least 130%, at least 135%, at least 140%,at least 145%, at least 150%, at least 160%, at least 170%, at least180%, at least 190%, or at least 200% for example, as compared to theability of bone marrow-derived dendritic cells to induceantigen-specific T cell proliferation in one or more bone marrow-deriveddendritic cells of a corresponding subject that is not treated with theagent. In other embodiments, the Siglec-9 agent, such as an antagonistanti-Siglec-9 antibody, may enhance and/or normalize the ability of bonemarrow-derived dendritic cells to induce antigen-specific T cellproliferation in one or more bone marrow-derived dendritic cells of asubject by at least at least 1.5 fold, at least 1.6 fold, at least 1.7fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, atleast 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, atleast 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold,at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0fold, at least 9.5 fold, or at least 10 fold, for example, as comparedto the ability of bone marrow-derived dendritic cells to induceantigen-specific T cell proliferation in one or more bone marrow-deriveddendritic cells of a corresponding subject that is not treated with theSiglec-9 agent.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with decreased or dysregulated ability of bone marrow-deriveddendritic cells to induce antigen-specific T cell proliferation,including without limitation, dementia, frontotemporal dementia,Alzheimer's disease, vascular dementia, mixed dementia,Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophiclateral sclerosis, Huntington's disease, taupathy disease, Nasu-Hakoladisease, stroke, acute trauma, chronic trauma, lupus, acute and chroniccolitis, rheumatoid arthritis, wound healing, Crohn's disease,inflammatory bowel disease, ulcerative colitis, obesity, malaria,essential tremor, central nervous system lupus, Behcet's disease,Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,Shy-Drager syndrome, progressive supranuclear palsy, cortical basalganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, and cancerincluding bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

Osteoclast Production

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may induceosteoclast production and/or increase the rate of osteoclastogenesisafter binding to a Siglec-9 protein expressed in a cell.

As used herein, an osteoclast is a type of bone cell that can removebone tissue by removing its mineralized matrix and breaking up theorganic bone (e.g., bone resorption). Osteoclasts can be formed by thefusion of cells of the myeloid lineage. In some embodiments, osteoclastsmay be characterized by high expression of tartrate resistant acidphosphatase (TRAP) and cathepsin K.

As used herein, the rate of osteoclastogenesis may be increased if therate of osteoclastogenesis in a subject treated with a Siglec-9 agent ofthe present disclosure, such as antagonist anti-Siglec-9 antibody, isgreater than the rate of osteoclastogenesis in a corresponding subjectthat is not treated with the Siglec-9 agent. In some embodiments, aSiglec-9 agent, such as an antagonist anti-Siglec-9 antibody of thepresent disclosure, may increase the rate of osteoclastogenesis in asubject by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 100%, atleast 110%, at least 115%, at least 120%, at least 125%, at least 130%,at least 135%, at least 140%, at least 145%, at least 150%, at least160%, at least 170%, at least 180%, at least 190%, or at least 200% forexample, as compared to rate of osteoclastogenesis in a correspondingsubject that is not treated with the Siglec-9 agent. In otherembodiments, a Siglec-9 agent, such as an antagonist anti-Siglec-9antibody of the present disclosure, may increase the rate ofosteoclastogenesis in a subject by at least 1.5 fold, at least 1.6 fold,at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, atleast 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, atleast 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold,at least 9.0 fold, at least 9.5 fold, or at least 10 fold, for example,as compared to rate of osteoclastogenesis in a corresponding subjectthat is not treated with the Siglec-9 agent.

As used herein, the rate of osteoclastogenesis may be decreased if therate of osteoclastogenesis in a subject treated with a Siglec-9 agent,such as an agonist anti-Siglec-9 antibody of the present disclosure, issmaller than the rate of osteoclastogenesis in a corresponding subjectthat is not treated with the Siglec-9 agent. In some embodiments, aSiglec-9 agent, such as an agonist anti-Siglec-9 antibody of the presentdisclosure, may decrease the rate of osteoclastogenesis in a subject byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 100%, at least 110%, atleast 115%, at least 120%, at least 125%, at least 130%, at least 135%,at least 140%, at least 145%, at least 150%, at least 160%, at least170%, at least 180%, at least 190%, or at least 200% for example, ascompared to rate of osteoclastogenesis in a corresponding subject thatis not treated with the Siglec-9 agent. In other embodiments, a Siglec-9agent, such as an agonist anti-Siglec-9 antibody of the presentdisclosure, may decrease the rate of osteoclastogenesis in a subject byat least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, atleast 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, atleast 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold,at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5fold, or at least 10 fold, for example, as compared to rate ofosteoclastogenesis in a corresponding subject that is not treated withthe Siglec-9 agent.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with abnormal bone formation and maintenance includingosteoporosis, which is associated with pathological decrease in bonedensity and osteoporotic diseases which are associated with pathologicalincrease in bone density.

Proliferation and Survival of Siglec-9-Expressing Cells

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may increase theproliferation, survival, and/or function of dendritic cells,macrophages, neutrophils, NK cells, monocytes, neutrophils, osteoclasts,Langerhans cells of skin, Kupffer cells, T cells, and microglial cellsafter binding to Siglec-9 protein expressed on a cell.

As used herein, macrophages of the present disclosure include, withoutlimitation, M1 macrophages, activated M1 macrophages, and M2macrophages. As used herein, neutrophils of the present disclosureinclude, without limitation, M1 neutrophils, activated M1 neutrophils,and M2 neutrophils. As used herein, natural killer (NK) cells of thepresent disclosure include, without limitation, M1 NK cells, activatedM1 NK cells, and M2 NK cells. As used herein, microglial cells of thepresent disclosure include, without limitation, M1 microglial cells,activated M1 microglial cells, and M2 microglial cells.

Microglial cells are a type of glial cell that are the residentmacrophages of the brain and spinal cord, and thus act as the first andmain form of active immune defense in the central nervous system (CNS).Microglial cells constitute 20% of the total glial cell populationwithin the brain. Microglial cells are constantly scavenging the CNS forplaques, damaged neurons and infectious agents. The brain and spinalcord are considered “immune privileged” organs in that they areseparated from the rest of the body by a series of endothelial cellsknown as the blood-brain barrier, which prevents most pathogens fromreaching the vulnerable nervous tissue. In the case where infectiousagents are directly introduced to the brain or cross the blood-brainbarrier, microglial cells must react quickly to limit inflammation anddestroy the infectious agents before they damage the sensitive neuraltissue. Due to the unavailability of antibodies from the rest of thebody (few antibodies are small enough to cross the blood brain barrier),microglia must be able to recognize foreign bodies, swallow them, andact as antigen-presenting cells activating T cells. Since this processmust be done quickly to prevent potentially fatal damage, microglialcells are extremely sensitive to even small pathological changes in theCNS. They achieve this sensitivity in part by having unique potassiumchannels that respond to even small changes in extracellular potassium.

In some embodiments, anti-Siglec-9 antibodies of the present disclosuremay increase the expression of CD80, CD83 and/or CD86 on dendriticcells, monocytes, macrophages, neutrophils, NK cells, and/or microglia.

As used herein, the rate of proliferation, survival, and/or function ofmacrophages, neutrophils, NK cells, dendritic cells, monocytes, T cells,and/or microglia may include increased expression if the rate ofproliferation, survival, and/or function of dendritic cells,macrophages, neutrophils, NK cells, monocytes, osteoclasts, Langerhanscells of skin, Kupffer cells, and/or microglia in a subject treated witha Siglec-9 agent, such as an anti-Siglec-9 antibody of the presentdisclosure, is greater than the rate of proliferation, survival, and/orfunction of dendritic cells, macrophages, neutrophils, NK cells,monocytes, osteoclasts, Langerhans cells of skin, Kupffer cells, Tcells, and/or microglia in a corresponding subject that is not treatedwith the Siglec-9 agent. In some embodiments, a Siglec-9 agent, such asan anti-Siglec-9 antibody of the present disclosure, may increase therate of proliferation, survival, and/or function of dendritic cells,macrophages, neutrophils, NK cells, monocytes, osteoclasts, Langerhanscells of skin, Kupffer cells, T cells, and/or microglia in a subject byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 100%, at least 110%, atleast 115%, at least 120%, at least 125%, at least 130%, at least 135%,at least 140%, at least 145%, at least 150%, at least 160%, at least170%, at least 180%, at least 190%, or at least 200% for example, ascompared to the rate of proliferation, survival, and/or function ofdendritic cells, macrophages, neutrophils, NK cells, monocytes,osteoclasts, Langerhans cells of skin, Kupffer cells, T cells, and/ormicroglia in a corresponding subject that is not treated with theSiglec-9 agent. In other embodiments, a Siglec-9 agent, such as ananti-Siglec-9 antibody of the present disclosure, may increase the rateof proliferation, survival, and/or function of dendritic cells,macrophages, neutrophils, NK cells, monocytes, osteoclasts, Langerhanscells of skin, Kupffer cells, T cells, and/or microglia in a subject byat least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, atleast 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, atleast 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold,at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5fold, or at least 10 fold, for example, as compared to the rate ofproliferation, survival, and/or function of dendritic cells,macrophages, neutrophils, NK cells, monocytes, osteoclasts, Langerhanscells of skin, Kupffer cells, T cells, and/or microglia in acorresponding subject that is not treated with the Siglec-9 agent.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with a reduction in proliferation, survival, increasedapoptosis and/or function of dendritic cells, neutrophils, macrophages,neutrophils, NK cells, monocytes, osteoclasts, Langerhans cells of skin,Kupffer cells, T cells, and/or microglia including without limitation,dementia, frontotemporal dementia, Alzheimer's disease, vasculardementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressurehydrocephalus, amyotrophic lateral sclerosis, Huntington's disease,taupathy disease, Nasu-Hakola disease, stroke, acute trauma, chronictrauma, lupus, acute and chronic colitis, rheumatoid arthritis, woundhealing, Crohn's disease, inflammatory bowel disease, ulcerativecolitis, obesity, malaria, essential tremor, central nervous systemlupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies,multiple system atrophy, Shy-Drager syndrome, progressive supranuclearpalsy, cortical basal ganglionic degeneration, acute disseminatedencephalomyelitis, granulomartous disorders, sarcoidosis, diseases ofaging, seizures, spinal cord injury, traumatic brain injury, age relatedmacular degeneration, glaucoma, retinitis pigmentosa, retinaldegeneration, respiratory tract infection, sepsis, eye infection,systemic infection, lupus, arthritis, multiple sclerosis, low bonedensity, osteoporosis, osteogenesis, osteopetrotic disease, Paget'sdisease of bone, and cancer including bladder cancer, brain cancer,breast cancer, colon cancer, rectal cancer, endometrial cancer, kidneycancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer,melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer,ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyeloid leukemia (CML), multiple myeloma, polycythemia vera, essentialthrombocytosis, primary or idiopathic myelofibrosis, primary oridiopathic myelosclerosis, myeloid-derived tumors, tumors that expressSiglec-9, thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

Clearance and Phagocytosis

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may induce clearanceand/or phagocytosis after binding to a Siglec-9 protein expressed in acell of one or more of apoptotic neurons, nerve tissue debris of thenervous system, non-nerve tissue debris of the nervous system,dysfunctional synapses, bacteria, other foreign bodies, disease-causingproteins, disease-causing peptides, disease-causing nucleic acid, ortumor cells. In certain embodiments, disease-causing proteins include,without limitation, amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides. In certain embodiments, disease-causing nucleicacids include, without limitation, antisense GGCCCC (G2C4)repeat-expansion RNA.

In some embodiments, the Siglec-9 agents of the present disclosure, suchas anti-Siglec-9 antibodies of the present disclosure, may induce of oneor more types of clearance, including without limitation, apoptoticneuron clearance, nerve tissue debris clearance, dysfunctional synapseclearance, non-nerve tissue debris clearance, bacteria or other foreignbody clearance, disease-causing protein clearance, disease-causingpeptide clearance, disease-causing nucleic acid clearance, and tumorcell clearance.

In some embodiments, the Siglec-9 agents of the present disclosure, suchas anti-Siglec-9 antibodies of the present disclosure, may inducephagocytosis of one or more of apoptotic neurons, nerve tissue debris,dysfunctional synapses, non-nerve tissue debris, bacteria, other foreignbodies, disease-causing proteins, disease-causing peptides,disease-causing nucleic acid, and/or tumor cells.

In some embodiments, the Siglec-9 agents of the present disclosure, suchas anti-Siglec-9 antibodies of the present disclosure, may increasephagocytosis by neutrophils, macrophages, neutrophils, NK cells,dendritic cells, monocytes, and/or microglia under conditions of reducedlevels of macrophage colony-stimulating factor (M-CSF).

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, are beneficial forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with apoptotic neurons, nerve tissue debris of the nervoussystem, non-nerve tissue debris of the nervous system, bacteria, otherforeign bodies, disease-causing proteins, including without limitation,dementia, frontotemporal dementia, Alzheimer's disease, vasculardementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressurehydrocephalus, amyotrophic lateral sclerosis, Huntington's disease,taupathy disease, Nasu-Hakola disease, stroke, acute trauma, chronictrauma, lupus, acute and chronic colitis, rheumatoid arthritis, woundhealing, Crohn's disease, inflammatory bowel disease, ulcerativecolitis, obesity, malaria, essential tremor, central nervous systemlupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies,multiple system atrophy, Shy-Drager syndrome, progressive supranuclearpalsy, cortical basal ganglionic degeneration, acute disseminatedencephalomyelitis, granulomartous disorders, sarcoidosis, diseases ofaging, seizures, spinal cord injury, traumatic brain injury, age relatedmacular degeneration, glaucoma, retinitis pigmentosa, retinaldegeneration, respiratory tract infection, sepsis, eye infection,systemic infection, lupus, arthritis, multiple sclerosis, low bonedensity, osteoporosis, osteogenesis, osteopetrotic disease, Paget'sdisease of bone, and cancer including bladder cancer, brain cancer,breast cancer, colon cancer, rectal cancer, endometrial cancer, kidneycancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer,melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer,ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyeloid leukemia (CML), multiple myeloma, polycythemia vera, essentialthrombocytosis, primary or idiopathic myelofibrosis, primary oridiopathic myelosclerosis, myeloid-derived tumors, tumors that expressSiglec-9, thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

Siglec-9-Dependent Gene Expression

In some embodiments, Siglec-9 agents of the present disclosure, such asantagonist anti-Siglec-9 antibodies of the present disclosure, maydecrease the activity and/or expression of Siglec-9-dependent genes, andby that increase gene expression associated with signaling cascade thatactivate the immune system such as gene expression associated with ITAMcontaining receptors, pattern recognition receptors, of Toll-likereceptors, of damage-associated molecular pattern (DAMP) receptors suchas one or more transcription factors of the nuclear factor of activatedT cells (NFAT) family of transcription factors.

In some embodiments, Siglec-9 agents of the present disclosure, such asantagonist anti-Siglec-9 antibodies of the present disclosure, arebeneficial for preventing, lowering the risk of, or treating conditionsand/or diseases associated with high levels of Siglec-9-dependent genes,including without limitation, dementia, frontotemporal dementia,Alzheimer's disease, vascular dementia, mixed dementia,Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophiclateral sclerosis, Huntington's disease, taupathy disease, Nasu-Hakoladisease, stroke, acute trauma, chronic trauma, lupus, acute and chroniccolitis, rheumatoid arthritis, wound healing, Crohn's disease,inflammatory bowel disease, ulcerative colitis, obesity, malaria,essential tremor, central nervous system lupus, Behcet's disease,Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,Shy-Drager syndrome, progressive supranuclear palsy, cortical basalganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, and cancerincluding bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and Haemophilus influenza.

Siglec-9-Dependent Activation of T Cells

In some embodiments, Siglec-9 agents of the present disclosure, such asantagonist anti-Siglec-9 antibodies of the present disclosure, mayincrease the activity of cytotoxic T cells helper T cells or both. Insome embodiments, Siglec-9 agents of the present disclosure, such asantagonist anti-Siglec-9 antibodies of the present disclosure, arebeneficial for preventing, lowering the risk of, or treating conditionsand/or diseases associated with decreased activity of cytotoxic T cellshelper T cells or both, including without limitation, tumors, includingsolid tumors such as bladder cancer, brain cancer, breast cancer, coloncancer, rectal cancer, endometrial cancer, kidney cancer, renal cellcancer, renal pelvis cancer, leukemia, lung cancer, melanoma,non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovariancancer, fibrosarcoma, and thyroid cancer.

Siglec-9-Dependent Inhibition of Neutrophils

In some embodiments, Siglec-9 agents of the present disclosure, such asagonist anti-Siglec-9 antibodies of the present disclosure, may decreasethe activity of neutrophils. In some embodiments, Siglec-9 agents of thepresent disclosure, such as agonist anti-Siglec-9 antibodies of thepresent disclosure, are beneficial for preventing, lowering the risk of,or treating conditions and/or diseases associated with decreasedactivity of the activity of natural killer cells, neutrophils or both,including without limitation, tumors, including solid tumors such asbladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer.

Siglec-9-Dependent Enhanced Cell Killing by Natural Killer (NK) Cells

In some embodiments, Siglec-9 agents of the present disclosure, such asantagonist anti-Siglec-9 antibodies of the present disclosure, mayincrease the killing activity of NK cells. In some embodiments, Siglec-9agents of the present disclosure, such as antagonistic anti-Siglec-9antibodies of the present disclosure, are beneficial for preventing,lowering the risk of, or treating conditions and/or diseases associatedwith decreased activity of natural killer cells, neutrophils or both,including without limitation, tumors, including solid tumors such asbladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer.

Siglec-9-Dependent Inhibition of Tumor-Associated Immune Cells

In some embodiments, Siglec-9 agents of the present disclosure, such asagonist anti-Siglec-9 antibodies of the present disclosure, may decreasethe activity, decrease the proliferation, decrease the survival,decrease the functionality, decrease infiltration to tumors or lymphoidorgans (e.g., the spleen and lymph nodes), and/or promote apoptosis ofT-regulatory cells or inhibitory tumor-imbedded immunosuppressordendritic cells or, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, or, myeloid-derived suppressorcells. In some embodiments, Siglec-9 agents of the present disclosure,such as agonist anti-Siglec-9 antibodies of the present disclosure, arebeneficial for preventing, lowering the risk of, or treating conditionsand/or diseases associated with the activity of one or more type ofimmune suppressor cells, including without limitation, tumors, includingsolid tumors that do not express Siglec-9 such as bladder cancer, braincancer, breast cancer, colon cancer, rectal cancer, endometrial cancer,kidney cancer, renal cell cancer, renal pelvis cancer, lung cancer,melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer,ovarian cancer, fibrosarcoma, thyroid cancer, and blood tumors thatexpress Siglec-9, such as leukemia cells.

Pharmaceutical Compositions

Siglec-9 agents of the present disclosure, such as anti-Siglec-9antibodies of the present disclosure, can be incorporated into a varietyof formulations for therapeutic administration by combining the agents,such as anti-Siglec-9 antibodies, with appropriate pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms. Examples of suchformulations include, without limitation, tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants,gels, microspheres, and aerosols. Pharmaceutical compositions caninclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers of diluents, which arevehicles commonly used to formulate pharmaceutical compositions foranimal or human administration. The diluent is selected so as not toaffect the biological activity of the combination. Examples of suchdiluents include, without limitation, distilled water, buffered water,physiological saline, PBS, Ringer's solution, dextrose solution, andHank's solution. A pharmaceutical composition or formulation of thepresent disclosure can further include other carriers, adjuvants, ornon-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients andthe like. The compositions can also include additional substances toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents, wetting agents and detergents.

A pharmaceutical composition of the present disclosure can also includeany of a variety of stabilizing agents, such as an antioxidant forexample. When the pharmaceutical composition includes a polypeptide, thepolypeptide can be complexed with various well-known compounds thatenhance the in vivo stability of the polypeptide, or otherwise enhanceits pharmacological properties (e.g., increase the half-life of thepolypeptide, reduce its toxicity, and enhance solubility or uptake).Examples of such modifications or complexing agents include, withoutlimitation, sulfate, gluconate, citrate and phosphate. The polypeptidesof a composition can also be complexed with molecules that enhance theirin vivo attributes. Such molecules include, without limitation,carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,sodium, potassium, calcium, magnesium, manganese), and lipids.

Further examples of formulations that are suitable for various types ofadministration can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.Examples of additional inactive ingredients that may be added to providedesirable color, taste, stability, buffering capacity, dispersion orother known desirable features are red iron oxide, silica gel, sodiumlauryl sulfate, titanium dioxide, and edible white ink. Similar diluentscan be used to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Formulations may be optimized for retention and stabilization in thebrain or central nervous system. When the agent is administered into thecranial compartment, it is desirable for the agent to be retained in thecompartment, and not to diffuse or otherwise cross the blood brainbarrier. Stabilization techniques include cross-linking, multimerizing,or linking to groups such as polyethylene glycol, polyacrylamide,neutral protein carriers, etc. in order to achieve an increase inmolecular weight.

Other strategies for increasing retention include the entrapment of anagent of the present disclosure, such as an anti-Siglec-9 antibody ofthe present disclosure, in a biodegradable or bioerodible implant. Therate of release of the therapeutically active agent is controlled by therate of transport through the polymeric matrix, and the biodegradationof the implant. The transport of drug through the polymer barrier willalso be affected by compound solubility, polymer hydrophilicity, extentof polymer cross-linking, expansion of the polymer upon water absorptionso as to make the polymer barrier more permeable to the drug, geometryof the implant, and the like. The implants are of dimensionscommensurate with the size and shape of the region selected as the siteof implantation. Implants may be particles, sheets, patches, plaques,fibers, microcapsules and the like and may be of any size or shapecompatible with the selected site of insertion.

The implants may be monolithic, i.e. having the active agenthomogenously distributed through the polymeric matrix, or encapsulated,where a reservoir of active agent is encapsulated by the polymericmatrix. The selection of the polymeric composition to be employed willvary with the site of administration, the desired period of treatment,patient tolerance, the nature of the disease to be treated and the like.Characteristics of the polymers will include biodegradability at thesite of implantation, compatibility with the agent of interest, ease ofencapsulation, a half-life in the physiological environment.

Biodegradable polymeric compositions which may be employed may beorganic esters or ethers, which when degraded result in physiologicallyacceptable degradation products, including the monomers. Anhydrides,amides, orthoesters or the like, by themselves or in combination withother monomers, may find use. The polymers will be condensationpolymers. The polymers may be cross-linked or non-cross-linked. Ofparticular interest are polymers of hydroxyaliphatic carboxylic acids,either homo- or copolymers, and polysaccharides. Included among thepolyesters of interest are polymers of D-lactic acid, L-lactic acid,racemic lactic acid, glycolic acid, polycaprolactone, and combinationsthereof. By employing the L-lactate or D-lactate, a slowly biodegradingpolymer is achieved, while degradation is substantially enhanced withthe racemate. Copolymers of glycolic and lactic acid are of particularinterest, where the rate of biodegradation is controlled by the ratio ofglycolic to lactic acid. The most rapidly degraded copolymer has roughlyequal amounts of glycolic and lactic acid, where either homopolymer ismore resistant to degradation. The ratio of glycolic acid to lactic acidwill also affect the brittleness of in the implant, where a moreflexible implant is desirable for larger geometries. Among thepolysaccharides of interest are calcium alginate, and functionalizedcelluloses, particularly carboxymethylcellulose esters characterized bybeing water insoluble, a molecular weight of about 5 kD to 500 kD, etc.Biodegradable hydrogels may also be employed in the implants of thepresent disclosure. Hydrogels are typically a copolymer material,characterized by the ability to imbibe a liquid. Exemplary biodegradablehydrogels which may be employed are described in Heller in: Hydrogels inMedicine and Pharmacy, N. A. Peppes ed., Vol. 1I1, CRC Press, BocaRaton, Fla., 1987, pp 137-149.

Pharmaceutical Dosages

Pharmaceutical compositions of the present disclosure containing aSiglec-9 agent of the present disclosure, such as an anti-Siglec-9antibody of the present disclosure, may be administered to an individualin need of treatment with the Siglec-9 agent, preferably a human, inaccord with known methods, such as intravenous administration as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, intracranial, intraspinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes.

Dosages and desired drug concentration of pharmaceutical compositions ofthe present disclosure may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary artisan. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles described in Mordenti, J. andChappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” InToxicokinetics and New Drug Development, Yacobi et al., Eds, PergamonPress, New York 1989, pp. 42-46.

For in vivo administration of any of the Siglec-9 agents of the presentdisclosure, such as any of the anti-Siglec-9 antibodies of the presentdisclosure, normal dosage amounts may vary from about 10 ng/kg up toabout 100 mg/kg of an individual's body weight or more per day,preferably about 1 mg/kg/day to 10 mg/kg/day, depending upon the routeof administration. For repeated administrations over several days orlonger, depending on the severity of the disease, disorder, or conditionto be treated, the treatment is sustained until a desired suppression ofsymptoms is achieved.

An exemplary dosing regimen may include administering an initial dose ofa Siglec-9 agent of the present disclosure, such as an anti-Siglec-9antibody, of about 2 mg/kg, followed by a weekly maintenance dose ofabout 1 mg/kg every other week. Other dosage regimens may be useful,depending on the pattern of pharmacokinetic decay that the physicianwishes to achieve. For example, dosing an individual from one totwenty-one times a week is contemplated herein. In certain embodiments,dosing ranging from about 3 μg/kg to about 2 mg/kg (such as about 3μg/kg, about 10 μg/kg, about 30 μg/kg, about 100 μg/kg, about 300 μg/kg,about 1 mg/kg, and about 2/mg/kg) may be used. In certain embodiments,dosing frequency is three times per day, twice per day, once per day,once every other day, once weekly, once every two weeks, once every fourweeks, once every five weeks, once every six weeks, once every sevenweeks, once every eight weeks, once every nine weeks, once every tenweeks, or once monthly, once every two months, once every three months,or longer. Progress of the therapy is easily monitored by conventionaltechniques and assays. The dosing regimen, including the Siglec-9 agent,such as the anti-Siglec-9 antibody administered, can vary over timeindependently of the dose used.

Dosages for a particular Siglec-9 agent, such as a particularanti-Siglec-9 antibody, may be determined empirically in individuals whohave been given one or more administrations of the Siglec-9 agent, suchas the anti-Siglec-9 antibody. Individuals are given incremental dosesof a Siglec-9 agent, such as an anti-Siglec-9 antibody. To assessefficacy of a Siglec-9 agent, such as an anti-Siglec-9 antibody, aclinical symptom of any of the diseases, disorders, or conditions of thepresent disclosure (e.g., frontotemporal dementia, Alzheimer's disease,vascular dementia, seizures, retinal dystrophy, a traumatic braininjury, a spinal cord injury, long-term depression, atheroscleroticvascular diseases, and undesirable symptoms of normal aging) can bemonitored.

Administration of a Siglec-9 agent, such as an anti-Siglec-9 antibody ofthe present disclosure, can be continuous or intermittent, depending,for example, on the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of a Siglec-9agent, such as an anti-Siglec-9 antibody, may be essentially continuousover a preselected period of time or may be in a series of spaced doses.

Guidance regarding particular dosages and methods of delivery isprovided in the literature; see, for example, U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212. It is within the scope of the presentdisclosure that different formulations will be effective for differenttreatments and different disorders, and that administration intended totreat a specific organ or tissue may necessitate delivery in a mannerdifferent from that to another organ or tissue. Moreover, dosages may beadministered by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

Therapeutic Uses

Further aspects of the present disclosure provide methods of modulating(e.g., activating or inhibiting) one or more Siglec-9 activities,including with limitation, modulating (e.g., activating or inhibiting) aSiglec-9 protein of the present disclosure, counteracting one or morephosphorylation of Tyr-433 and Tyr-456 by a Src family tyrosine kinase,such as Syk, LCK, FYM, and/or ZAP70; recruitment of and binding to thetyrosine-specific protein phosphatases SHP1 and SHP2; recruitment of andbinding to PLC-gamma1, which acts as a guanine nucleotide exchangefactor for Dynamini-1; recruitment of and binding to SH2-domaincontaining protein (e.g., Crk1); recruitment of and binding to thespleen tyrosine kinase Syk; recruitment of and binding to SH3-SH2-SH3growth factor receptor-bound protein 2 (Grb2); recruitment of andbinding to multiple SH2-containing proteins; modulating (e.g.,activating or inhibiting) expression of one or more pro-inflammatorycytokines, optionally wherein the one or more anti-inflammatorycytokines are selected from FN-α4, IFN-beta, IL-113, IL-1alpha, TNF-α,IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ, OSM, CNTF, GM-CSF,IL-11, IL-12, IL-17, IL-18, IL-33, MCP-1, and MIP-1-beta; modulating(e.g., activating or inhibiting) expression of one or morepro-inflammatory cytokines in one or more cells selected frommacrophages, neutrophils, NK cells, dendritic cells, bone marrow-deriveddendritic cells, monocytes, osteoclasts, T cells, T helper cells,cytotoxic T cells, granulocytes, and microglial cells; modulating (e.g.,activating or inhibiting) expression of one or more anti-inflammatorycytokines, optionally wherein the one or more anti-inflammatorycytokines are selected from IL-4, IL-10, IL-13, IL-35, IL-16, TGF-beta,IL-1Rα, G-CSF, and soluble receptors for TNF, IFN-beta1a, IFN-beta1b, orIL-6; modulating (e.g., activating or inhibiting) expression of one ormore anti-inflammatory cytokines in one or more cells selected frommacrophages, neutrophils, NK cells, dendritic cells, bone marrow-deriveddendritic cells, monocytes, osteoclasts, T cells, T helper cells,cytotoxic T cells, granulocytes, and microglial cells; modulating (e.g.,activating or inhibiting) expression of one or more proteins selectedfrom C1qa, C1qB, C1qC, C1s, C1R, C4, C2, C3, ITGB2, HMOX1, LAT2, CASP1,CSTA, VSIG4, MS4A4A, C3AR1, GPX1, TyroBP, ALOX5AP, ITGAM, SLC7A7, CD4,ITGAX, and PYCARD; activation of extracellular signal-regulated kinase(ERK) phosphorylation; modulating (e.g., activating or inhibiting)tyrosine phosphorylation on one or more cellular proteins, optionally,wherein the one or more cellular proteins comprise ZAP-70 and thetyrosine phosphorylation occurs on Tyr-319 of ZAP-70; modulating (e.g.,activating or inhibiting) expression of C—C chemokine receptor 7 (CCR7);activation of microglial cell chemotaxis toward CCL19-expressing andCCL21-expressing cells; modulating (e.g., activating or inhibiting) Tcell proliferation induced by one or more cells selected from dendriticcells, bone marrow-derived dendritic cells, monocytes, microglia, M1microglia, activated M1 microglia, M2 microglia, macrophages,neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells,activated M1 macrophages, activated M1 neutrophils, activated M1 NKcells, M2 macrophages, M2 neutrophils, and M2 NK cells; modulating(e.g., activating or inhibiting) osteoclast production, modulating(e.g., activating or inhibiting) rate of osteoclastogenesis, or both;modulating (e.g., activating or inhibiting) survival of one or morecells selected from dendritic cells, bone marrow-derived dendriticcells, macrophages, neutrophils, NK cells, M1 macrophages, M1neutrophils, M1 NK cells, activated M1 macrophages, activated M1neutrophils, activated M1 NK cells, M2 macrophages, M2 neutrophils, M2NK cells, monocytes, osteoclasts, T cells, T helper cells, cytotoxic Tcells, granulocytes, neutrophils, microglia, M1 microglia, activated M1microglia, and M2 microglia; modulating (e.g., activating or inhibiting)proliferation of one or more cells selected from dendritic cells, bonemarrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages,activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helpercells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1microglia, activated M1 microglia, and M2 microglia; modulating (e.g.,activating or inhibiting) migration of one or more cells selected fromdendritic cells, bone marrow-derived dendritic cells, macrophages,neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells,activated M1 macrophages, activated M1 neutrophils, activated M1 NKcells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; modulating (e.g., activating or inhibiting) one or morefunctions of one or more cells selected from dendritic cells, bonemarrow-derived dendritic cells, macrophages, neutrophils, NK cells, M1macrophages, M1 neutrophils, M1 NK cells, activated M1 macrophages,activated M1 neutrophils, activated M1 NK cells, M2 macrophages, M2neutrophils, M2 NK cells, monocytes, osteoclasts, T cells, T helpercells, cytotoxic T cells, granulocytes, neutrophils, microglia, M1microglia, activated M1 microglia, and M2 microglia; modulating (e.g.,activating or inhibiting) maturation of one or more cells selected fromdendritic cells, bone marrow-derived dendritic cells, macrophages,neutrophils, NK cells, M1 macrophages, M1 neutrophils, M1 NK cells,activated M1 macrophages, activated M1 neutrophils, activated M1 NKcells, M2 macrophages, M2 neutrophils, M2 NK cells, monocytes,osteoclasts, T cells, T helper cells, cytotoxic T cells, granulocytes,neutrophils, microglia, M1 microglia, activated M1 microglia, and M2microglia; activation of one or more types of clearance selected fromapoptotic neuron clearance, nerve tissue debris clearance, dysfunctionalsynapse clearance, non-nerve tissue debris clearance, bacteriaclearance, other foreign body clearance, disease-causing proteinclearance, disease-causing peptide clearance, and tumor cell clearance;optionally wherein the disease-causing protein is selected from amyloidbeta, oligomeric amyloid beta, amyloid beta plaques, amyloid precursorprotein or fragments thereof, Tau, TAPP, alpha-synuclein, TDP-43, FUSprotein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein,prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase,ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10,Lewy body, atrial natriuretic factor, islet amyloid polypeptide,insulin, apolipoprotein AI, serum amyloid A, medin, prolactin,transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides and the tumor cell is from a cancer selected frombladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer; activation of phagocytosis of one or more of apoptoticneurons, nerve tissue debris, dysfunctional synapses non-nerve tissuedebris, bacteria, other foreign bodies, disease-causing proteins,disease-causing peptides, disease-causing nucleic acids, or tumor cells;optionally wherein the disease-causing nucleic acids are antisenseGGCCCC (G2C4) repeat-expansion RNA, the disease-causing proteins areselected from amyloid beta, oligomeric amyloid beta, amyloid betaplaques, amyloid precursor protein or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open readingframe 72), c9RAN protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, ataxin 1, ataxin 2, ataxin 3, ataxin 7,ataxin 8, ataxin 10, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine(PR) repeat peptides, and the tumor cells are from a cancer selectedfrom bladder cancer, brain cancer, breast cancer, colon cancer, rectalcancer, endometrial cancer, kidney cancer, renal cell cancer, renalpelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, orthyroid cancer; inhibiting binding to Siglec-9 ligand on tumor cells;modulating (e.g., activating or inhibiting) binding to Siglec-9 ligandon cells selected from neutrophils, dendritic cells, bone marrow-deriveddendritic cells, monocytes, microglia, macrophages, and NK cells;activation of tumor cell killing by one or more of microglia,macrophages, neutrophils, NK cells, dendritic cells, bone marrow-deriveddendritic cells, neutrophils, T cells, T helper cells, or cytotoxic Tcells; activating anti-tumor cell proliferation activity of one or moreof microglia, macrophages, neutrophils, NK cells, dendritic cells, bonemarrow-derived dendritic cells, neutrophils, T cells, T helper cells, orcytotoxic T cells; activation of anti-tumor cell metastasis activity ofone or more of microglia, macrophages, neutrophils, NK cells, dendriticcells, bone marrow-derived dendritic cells, neutrophils, T cells, Thelper cells, or cytotoxic T cells; modulating (e.g., activating orinhibiting) of one or more ITAM motif containing receptors, optionallywherein the one or more ITAM motif containing receptors are selectedfrom TREM1, TREM2, Sirp beta, FcgR, DAP10, and DAP12; modulating (e.g.,activating or inhibiting) of signaling by one or more patternrecognition receptors (PRRs), optionally wherein the one or more PRRsare selected from receptors that identify pathogen-associated molecularpatterns (PAMPs), receptors that identify damage-associated molecularpatterns (DAMPs), and any combination thereof; modulating (e.g.,activating or inhibiting) of one or more receptors comprising the motifD/Ex₀₋₂YxxL/IX₆₋₈YxxL/I (SEQ ID NO: 252); modulating (e.g., activatingor inhibiting) of signaling by one or more Toll-like receptors;modulating (e.g., activating or inhibiting) of the JAK-STAT signalingpathway; modulating (e.g., activating or inhibiting) of nuclear factorkappa-light-chain-enhancer of activated B cells (NFκB); phosphorylationof an ITAM motif containing receptor; modulating (e.g., activating orinhibiting) expression of one or more inflammatory receptors, proteinsof the complement cascade, and/or receptors, optionally wherein the oneor more inflammatory receptors, proteins of the complement cascade,and/or receptors comprise CD86, C1qa, C1qB, C1qC, C1s, C1R, C4, C2, C3,ITGB2, HMOX1, LAT2, CASP1, CSTA, VSIG4, MS4A4A, C3AR1, GPX1, TyroBP,ALOX5AP, ITGAM, SLC7A7, CD4, ITGAX, and/or PYCARD, and the one or moreinflammatory receptors, proteins of the complement cascade, and/orreceptors are expressed on one or more of microglia, macrophages,neutrophils, NK cells, dendritic cells, bone marrow-derived dendriticcells, neutrophils, T cells, T helper cells, or cytotoxic T cells;modulating (e.g., activating or inhibiting) expression of one or moreSiglec-9-dependent genes; normalization of disrupted Siglec-9-dependentgene expression; modulating (e.g., activating or inhibiting) expressionof one or more ITAM-dependent genes, optionally wherein the one moreITAM-dependent genes are activated by nuclear factor of activated Tcells (NFAT) transcription factors; rescuing functionality of one ormore of immunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, and regulatory T cells; reducinginfiltration of one or more of immunosuppressor dendritic cells,immunosuppressor macrophages, immunosuppressor neutrophils,immunosuppressor NK cells, myeloid-derived suppressor cells,tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, and regulatory T cells into tumors;increasing the number of tumor-promoting myeloid/granulocyticimmune-suppressive cells in a tumor, in peripheral blood, or otherlymphoid organ; enhancing tumor-promoting activity of myeloid-derivedsuppressor cells; increasing expression of tumor-promoting cytokines ina tumor or in peripheral blood, optionally wherein the tumor-promotingcytokines are TGF-beta or IL-10; increasing tumor infiltration oftumor-promoting FoxP3+ regulatory T lymphocytes; enhancingtumor-promoting activity of myeloid-derived suppressor cells (MDSC);decreasing activation of tumor-specific T lymphocytes with tumor killingpotential; decreasing infiltration of tumor-specific NK cells with tumorkilling potential; decreasing the tumor killing potential of NK cells;decreasing infiltration of tumor-specific B lymphocytes with potentialto enhance immune response; decreasing infiltration of tumor-specific Tlymphocytes with tumor killing potential; increasing tumor volume;increasing tumor growth rate; increasing metastasis; increasing rate oftumor recurrence; decreasing efficacy of one or more immune-therapiesthat modulate anti-tumor T cell responses, optionally wherein the one ormore immune-therapies are immune-therapies that target one or moretarget proteins selected from PD1/PDL1, CD40, OX40, ICOS, CD28,CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM,LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR,LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1 receptor, and anycombination thereof, or of one or more cancer vaccines; inhibition ofPLCγ/PKC/calcium mobilization; and/or inhibition of PI3K/Akt, Ras/MAPKsignaling in an individual in need thereof, by administering to theindividual a therapeutically effective amount of a Siglec-9 agent of thepresent disclosure, such as an anti-Siglec-9 antibody of the presentdisclosure, to modulate (e.g., activate or inhibit) one or more of theSiglec-9 activities in the individual.

As disclosed herein, Siglec-9 agents of the present disclosure that bindSiglec-9, decrease cellular levels of Siglec-9, inhibit interactionbetween Siglec-9 and one or more Siglec-9 ligands, or any combinationthereof, such as anti-Siglec-9 antibodies of the present disclosure, maybe used for preventing, reducing risk, or treating dementia,frontotemporal dementia, Alzheimer's disease, vascular dementia, mixeddementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus,amyotrophic lateral sclerosis, Huntington's disease, taupathy disease,Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acuteand chronic colitis, rheumatoid arthritis, wound healing, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, obesity,malaria, essential tremor, central nervous system lupus, Behcet'sdisease, Parkinson's disease, dementia with Lewy bodies, multiple systematrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticalbasal ganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, and cancerincluding bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and/or Haemophilus influenza. In someembodiments, the agents are selected from antibodies, soluble Siglec-9receptors, Siglec-9-Fc fusion proteins, Siglec-9 immunoadhesins, solubleSiglec receptors that binds one or more Siglec-9 ligands, Siglec-Fcfusion proteins, Siglec immunoadhesins, antisense molecules, siRNAs,small molecule inhibitors, proteins, and peptides. In some embodiments,the Siglec-9 agents are agonist antibodies. In some embodiments, theSiglec-9 agents are inert antibodies. In some embodiments, the Siglec-9agents are antagonist antibodies.

In some embodiments, the present disclosure provides methods ofpreventing, reducing risk, or treating dementia, frontotemporaldementia, Alzheimer's disease, vascular dementia, mixed dementia,Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophiclateral sclerosis, Huntington's disease, taupathy disease, Nasu-Hakoladisease, stroke, acute trauma, chronic trauma, lupus, acute and chroniccolitis, rheumatoid arthritis, wound healing, Crohn's disease,inflammatory bowel disease, ulcerative colitis, obesity, malaria,essential tremor, central nervous system lupus, Behcet's disease,Parkinson's disease, dementia with Lewy bodies, multiple system atrophy,Shy-Drager syndrome, progressive supranuclear palsy, cortical basalganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, andcancer, bladder cancer, brain cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, renal cell cancer,renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin'slymphoma, pancreatic cancer, prostate cancer, ovarian cancer,fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia(CML), multiple myeloma, polycythemia vera, essential thrombocytosis,primary or idiopathic myelofibrosis, primary or idiopathicmyelosclerosis, myeloid-derived tumors, tumors that express Siglec-9,thyroid cancer, infections, CNS herpes, parasitic infections,Trypanosome infection, Cruzi infection, Pseudomonas aeruginosainfection, Leishmania donovani infection, group B Streptococcusinfection, Campylobacter jejuni infection, Neisseria meningiditisinfection, type I HIV, and/or Haemophilus influenza, by administering toan individual in need thereof a therapeutically effective amount of anagent of the present disclosure that decreases cellular levels ofSiglec-9, inhibits interaction between Siglec-9 and one or more Siglec-9ligands, or both. In some embodiments, the agent is selected from anantibody, a soluble Siglec-9 receptor, a Siglec-9-Fc fusion protein, aSiglec-9 immunoadhesin, a soluble Siglec receptor that binds one or moreSiglec-9 ligands, a Siglec-Fc fusion protein, a Siglec immunoadhesin, anantisense molecule, an siRNA, a small molecule inhibitor, a protein, anda peptide. In some embodiments, the agent is an anti-Siglec-9 antibodyof the present disclosure.

In some embodiments, the present disclosure provides methods ofpreventing, reducing risk, or treating cancer, by administering to anindividual in need thereof, a therapeutically effective amount of anagent of the present disclosure that decreases cellular levels ofSiglec-9, inhibits interaction between Siglec-9 and one or more Siglec-9ligands, or both. In some embodiments, the agent is selected from anantibody, a soluble Siglec-9 receptor, a Siglec-9-Fc fusion protein, aSiglec-9 immunoadhesin, a soluble Siglec receptor that binds one or moreSiglec-9 ligands, a Siglec-Fc fusion protein, a Siglec immunoadhesin, anantisense molecule, an siRNA, a small molecule inhibitor, a protein, anda peptide. In certain embodiments, the agent is an anti-Siglec-9antibody of the present disclosure. In some embodiments, theanti-Siglec-9 antibody inhibits one or more Siglec-9 activities selectedfrom: (a) promoting proliferation, maturation, migration,differentiation, and/or functionality of one or more of immunosuppressordendritic cells, immunosuppressor macrophages, immunosuppressorneutrophils, immunosuppressor NK cells, myeloid derived suppressorcells, tumor-associated macrophages, tumor-associated suppressorneutrophils, tumor-associated suppressor NK cells, and regulatory Tcells; (b) enhancing infiltration of one or more of immunosuppressordendritic cells, immunosuppressor macrophages, immunosuppressorneutrophils, immunosuppressor NK cells, myeloid derived suppressorcells, tumor-associated macrophages, tumor-associated suppressorneutrophils, tumor-associated suppressor NK cells, and regulatory Tcells into tumors; (c) increasing number of tumor-promotingmyeloid/granulocytic immune-suppressive cells in a tumor, in peripheralblood, or other lymphoid organ; (d) enhancing tumor-promoting activityof myeloid-derived suppressor cells (MDSC); (e) increasing expression oftumor-promoting cytokines in a tumor or in peripheral blood, optionallywherein the tumor-promoting cytokines are TGF-beta or IL-10; (f)increasing tumor infiltration of tumor-promoting FoxP3+ regulatory Tlymphocytes; (g) decreasing activation of tumor-specific T lymphocyteswith tumor killing potential; (h) decreasing infiltration oftumor-specific T lymphocytes with tumor killing potential; (i)decreasing infiltration of tumor-specific NK cells with tumor killingpotential; (j) decreasing the tumor killing potential of NK cells; (k)decreasing infiltration of tumor-specific B lymphocytes with potentialto enhance immune response; (l) increasing tumor volume; (m) increasingtumor growth rate; (n) increasing metastasis; (o) increasing rate oftumor recurrence; (p) decreasing efficacy of one or moreimmune-therapies that modulate anti-tumor T cell responses, optionallywherein the one or more immune-therapies are immune-therapies thattarget one or more target proteins selected from PD1/PDL1, CD40, OX40,ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3,B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3,TIM4, A2AR, LAG3, DR-5, CD2, CD5, TREM1, TREM2, CD39, CD73, CSF-1receptor, and any combination thereof, or cancer vaccines; (q)inhibition of PLCγ/PKC/calcium mobilization; and (r) inhibition ofPI3K/Akt, Ras/MAPK signaling. In some embodiments, the anti-Siglec-9antibody inhibits one or more Siglec-9 activities selected from thegroup consisting of: (a) promoting proliferation, maturation, migration,differentiation, and/or functionality of one or more of immunosuppressordendritic cells, immunosuppressor macrophages, immunosuppressorneutrophils, non-tumorigenic myeloid derived suppressor cells,tumor-associated macrophages, non-tumorigenic CD14⁺ myeloid cells, andregulatory T cells; (b) enhancing infiltration of one or more ofimmunosuppressor dendritic cells, immunosuppressor macrophages,immunosuppressor neutrophils, non-tumorigenic myeloid derived suppressorcells, tumor-associated macrophages, and regulatory T cells into tumors;(c) increasing number of tumor-promoting myeloid/granulocyticimmune-suppressive cells and/or non-tumorigenic CD14⁺ myeloid cells in atumor, in peripheral blood, or other lymphoid organ; (d) enhancingtumor-promoting activity of non-tumorigenic myeloid-derived suppressorcells and/or non-tumorigenic CD14⁺ myeloid cells; (e) increasingexpression of tumor-promoting cytokines in a tumor or in peripheralblood, optionally wherein the tumor-promoting cytokines are TGF-beta orIL-10; (f) increasing tumor infiltration of tumor-promoting FoxP3+regulatory T lymphocytes; (g) decreasing activation of tumor-specific Tlymphocytes with tumor killing potential; (h) decreasing infiltration oftumor-specific T lymphocytes with tumor killing potential; (i)decreasing infiltration of tumor-specific NK cells with tumor killingpotential; (j) decreasing tumor killing potential of NK cells; (k)decreasing infiltration of tumor-specific B lymphocytes with potentialto enhance immune response; (1) increasing tumor volume; (m) increasingtumor growth rate; (n) increasing metastasis; (o) increasing rate oftumor recurrence; (p) increasing expression of one or more PD-1 ligands;(q) decreasing efficacy of one or more immune-therapies that modulateanti-tumor T cell responses, optionally wherein the one or moreimmune-therapies are immune-therapies that target one or more proteinsselected from the group consisting of CD40, OX40, ICOS, CD28,CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM,LIGHT, BTLA, KIR, GAL9, CD2, CD5, CD39, CD73, CD30, TIGIT, VISTA, TIM1,TIM3, TIM4, A2AR, LAG, DR-5, TREM1, TREM2, CSF-1 receptor, and anycombination thereof, or of one or more cancer vaccines; (r) inhibitionof PLCγ/PKC/calcium mobilization; (s) inhibition of PI3K/Akt, Ras/MAPKsignaling; and (t) decreasing efficacy of one or more chemotherapyagents, optionally wherein the one or more of the chemotherapy agentsare gemcitabine, capecitabine, anthracyclines, doxorubicin(Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel (Taxol®),docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide(Cytoxan®), carboplatin (Paraplatin®), and any combination thereof. Insome embodiments, the anti-Siglec-9 antibody exhibits one or moreactivities selected from the group consisting of consisting of: (a)increasing the number of tumor infiltrating CD3⁺ T cells; (b) decreasingcellular levels of Siglec-9 in non-tumorigenic CD14+ myeloid cells,optionally wherein the non-tumorigenic CD14⁺ myeloid cells are tumorinfiltrating cells or optionally wherein the non-tumorigenic CD14⁺myeloid cells are present in blood; (c) reducing the number ofnon-tumorigenic CD14⁺ myeloid cells, optionally wherein thenon-tumorigenic CD14⁺ myeloid cells are tumor infiltrating cells oroptionally wherein the non-tumorigenic CD14⁺ myeloid cells are presentin blood; (d) reducing PD-L1 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (e) reducing PD-L2 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (f) reducing B7-H2 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (g) reducingB7-H3 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (h)reducing CD200R levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (i) reducing CD163 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (j) reducing CD206 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (k) decreasing tumor growthrate of solid tumors; (l) reducing tumor volume; (m) increasing efficacyof one or more PD-1 inhibitors; (n) increasing efficacy of one or morecheckpoint inhibitor therapies and/or immune-modulating therapies,optionally wherein the one or more checkpoint inhibitor therapies and/orimmune-modulating therapies target one or more of CTLA4, the adenosinepathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; (o)increasing efficacy of one or more chemotherapy agents, optionallywherein the one or more of the chemotherapy agents are gemcitabine,capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin(Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®),5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin(Paraplatin®), and any combination thereof; (p) increasing proliferationof T cells in the presence of non-tumorigenic myeloid-derived suppressorcells (MDSC); and (q) inhibiting differentiation, survival, and/or oneor more functions of non-tumorigenic myeloid-derived suppressor cells(MDSC); and (r) killing Siglec-9-expressing immunosuppressor myeloidcells and/or CD14-expressing cells in solid tumors and associated bloodvessels when conjugated to a chemical or radioactive toxin. In someembodiments that may be combined with any of the preceding embodiments,the disease, disorder, or injury is cancer, and the agent exhibits oneor more activities selected from the group consisting of consisting of:(a) increasing the number of tumor infiltrating CD3⁺ T cells; (b)decreasing cellular levels of Siglec-9 in non-tumorigenic CD14+ myeloidcells, optionally wherein the non-tumorigenic CD14⁺ myeloid cells aretumor infiltrating cells or optionally wherein the non-tumorigenic CD14⁺myeloid cells are present in blood; (c) reducing the number ofnon-tumorigenic CD14⁺ myeloid cells, optionally wherein thenon-tumorigenic CD14⁺ myeloid cells are tumor infiltrating cells oroptionally wherein the non-tumorigenic CD14⁺ myeloid cells are presentin blood; (d) reducing PD-L1 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (e) reducing PD-L2 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (f) reducing B7-H2 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (g) reducingB7-H3 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (h)reducing CD200R levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (i) reducing CD163 levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (j) reducing CD206 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (k) decreasing tumor growthrate of solid tumors; (l) reducing tumor volume; (m) increasing efficacyof one or more PD-1 inhibitors; (n) increasing efficacy of one or morecheckpoint inhibitor therapies and/or immune-modulating therapies,optionally wherein the one or more checkpoint inhibitor therapies and/orimmune-modulating therapies target one or more of CTLA4, the adenosinepathway, PD-L1, PD-L2, OX40, TIM3, LAG3, or any combination thereof; (o)increasing efficacy of one or more chemotherapy agents, optionallywherein the one or more of the chemotherapy agents are gemcitabine,capecitabine, anthracyclines, doxorubicin (Adriamycin®), epirubicin(Ellence®), taxanes, paclitaxel (Taxol®), docetaxel (Taxotere®),5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin(Paraplatin®), and any combination thereof; (p) increasing proliferationof T cells in the presence of non-tumorigenic myeloid-derived suppressorcells (MDSC); and (q) inhibiting differentiation, survival, and/or oneor more functions of non-tumorigenic myeloid-derived suppressor cells(MDSC); and (r) killing Siglec-9-expressing immunosuppressor myeloidcells and/or CD14-expressing cells in solid tumors and associated bloodvessels when conjugated to a chemical or radioactive toxin. In someembodiments that may be combined with any of the preceding embodiments,the agent exhibits one or more activities selected from the groupconsisting of consisting of: (a) increasing the number of tumorinfiltrating CD3⁺ T cells; (b) decreasing cellular levels of Siglec-9 innon-tumorigenic CD14+ myeloid cells, optionally wherein thenon-tumorigenic CD14⁺ myeloid cells are tumor infiltrating cells oroptionally wherein the non-tumorigenic CD14⁺ myeloid cells are presentin blood; (c) reducing the number of non-tumorigenic CD14⁺ myeloidcells, optionally wherein the non-tumorigenic CD14⁺ myeloid cells aretumor infiltrating cells or optionally wherein the non-tumorigenic CD14⁺myeloid cells are present in blood; (d) reducing PD-L1 levels in one ormore cells, optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (e) reducing PD-L2 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (f) reducingB7-H2 levels in one or more cells, optionally wherein the one or morecells are non-tumorigenic myeloid-derived suppressor cells (MDSC); (g)reducing B7-H3 levels in one or more cells, optionally wherein the oneor more cells are non-tumorigenic myeloid-derived suppressor cells(MDSC); (h) reducing CD200R levels in one or more cells, optionallywherein the one or more cells are non-tumorigenic myeloid-derivedsuppressor cells (MDSC); (i) reducing CD163 levels in one or more cells,optionally wherein the one or more cells are non-tumorigenicmyeloid-derived suppressor cells (MDSC); (j) reducing CD206 levels inone or more cells, optionally wherein the one or more cells arenon-tumorigenic myeloid-derived suppressor cells (MDSC); (k) decreasingtumor growth rate of solid tumors; (l) reducing tumor volume; (m)increasing efficacy of one or more PD-1 inhibitors; (n) increasingefficacy of one or more checkpoint inhibitor therapies and/orimmune-modulating therapies, optionally wherein the one or morecheckpoint inhibitor therapies and/or immune-modulating therapies targetone or more of CTLA4, the adenosine pathway, PD-L1, PD-L2, OX40, TIM3,LAG3, or any combination thereof; (o) increasing efficacy of one or morechemotherapy agents, optionally wherein the one or more of thechemotherapy agents are gemcitabine, capecitabine, anthracyclines,doxorubicin (Adriamycin®), epirubicin (Ellence®), taxanes, paclitaxel(Taxol®), docetaxel (Taxotere®), 5-fluorouracil (5-FU), cyclophosphamide(Cytoxan®), carboplatin (Paraplatin®), and any combination thereof; (p)increasing proliferation of T cells in the presence of non-tumorigenicmyeloid-derived suppressor cells (MDSC); and (q) inhibitingdifferentiation, survival, and/or one or more functions ofnon-tumorigenic myeloid-derived suppressor cells (MDSC); and (r) killingSiglec-9-expressing immunosuppressor myeloid cells and/orCD14-expressing cells in solid tumors and associated blood vessels whenconjugated to a chemical or radioactive toxin.

As disclosed herein, agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may also be used forinducing and/or promoting the survival maturation, functionality,migration, or proliferation of one or more immune cells (e.g., innateimmune cells or adaptive immune cells). In some embodiments, the presentdisclosure provides methods of inducing or promoting the survival,maturation, functionality, migration, or proliferation of one or moreimmune cells in an individual in need thereof, by administering to theindividual a therapeutically effective amount of an agent of the presentdisclosure that decreases cellular levels of Siglec-9, inhibitsinteraction between Siglec-9 and one or more Siglec-9 ligands, or both.In some embodiments, the agent is selected from an antibody, a solubleSiglec-9 receptor, a Siglec-9-Fc fusion protein, a Siglec-9immunoadhesin, a soluble Siglec receptor that binds one or more Siglec-9ligands, a Siglec-Fc fusion protein, a Siglec immunoadhesin, anantisense molecule, an siRNA, a small molecule inhibitor, a protein, anda peptide. In some embodiments, the agent is an isolated anti-Siglec-9antibody of the present disclosure. In some embodiments, the one or moreimmune cells are selected from dendritic cells, macrophages,neutrophils, NK cells, microglia, T cells, T helper cells, cytotoxic Tcells, and any combination thereof.

Other aspects of the present disclosure relate to a method of assessingresponsiveness of a subject in need thereof to an agent that binds orinteracts with Siglec-9, the method comprising: a. measuring theexpression levels of CD45⁺ and CD14⁺ on non-tumorigenic myeloid cells ina blood sample obtained from the subject prior to administering to thesubject an anti-Siglec-9 antibody; b. administering to the subject atherapeutically effective amount of the agent; and c. measuring theexpression levels of CD45⁺ and CD14⁺ on non-tumorigenic myeloid cells ina blood sample obtained from the subject after administration of theanti-Siglec-9 antibody, wherein a reduction in the levels of CD45⁺CD14⁺on non-tumorigenic myeloid cells after administration of theanti-Siglec-9 antibody indicates the subject is responsive to the agent.In some embodiments, the method of assessing responsiveness furthercomprises administering one or more additional therapeutically effectiveamounts of the agent. In some embodiments that may be combined with anyof the preceding embodiments, the agent is selected from the groupconsisting of an antibody, a soluble Siglec-9 receptor, a Siglec-9-Fcfusion protein, a Siglec-9 immunoadhesin, a soluble Siglec receptor, aSiglec-Fc fusion protein, a Siglec immunoadhesin, an antisense molecule,an siRNA, a small molecule inhibitor, a protein, and a peptide. Anysuitable methods for obtaining a sample, such as a blood sample, may beused. In some embodiments, the method of assessing responsivenessfurther comprises administering one or more additional therapeuticallyeffective amounts of the agent. In some embodiments, the agent isselected from the group consisting of an antibody, a soluble Siglec-9receptor, a Siglec-9-Fc fusion protein, a Siglec-9 immunoadhesin, asoluble Siglec receptor, a Siglec-Fc fusion protein, a Siglecimmunoadhesin, an antisense molecule, an siRNA, a small moleculeinhibitor, a protein, and a peptide. In some embodiments, the agent isan isolated anti-Siglec-9 antibody or anti-Siglec-9 antibody conjugate.In some embodiments, the anti-Siglec-9 antibody is the anti-Siglec-9antibody of the present disclosure. In some embodiments, the subject ishuman.

In some embodiments, the agent is an agonist anti-Siglec-9 antibody. Insome embodiments, the agent is a transient agonist anti-Siglec-9antibody of the present disclosure that initially acts as an agonist andthen acts as a long-term antagonist antibody. In some embodiments, theagent is an inert anti-Siglec-9 antibody. In some embodiments, the agentis an antagonist anti-Siglec-9 antibody. In some embodiments, theanti-Siglec-9 antibody reduces cellular (e.g., cell surface,intracellular, or total) levels of Siglec-9. In some embodiments, theanti-Siglec-9 antibody induces degradation of Siglec-9. In someembodiments, the anti-Siglec-9 antibody induces cleavage of Siglec-9. Insome embodiments, the anti-Siglec-9 antibody induces internalization ofSiglec-9. In some embodiments, the anti-Siglec-9 antibody inducesshedding of Siglec-9. In some embodiments, the anti-Siglec-9 antibodyinduces downregulation of Siglec-9 expression. In some embodiments, theanti-Siglec-9 antibody inhibits interaction (e.g., binding) betweenSiglec-9 and one or more Siglec-9 ligands. In some embodiments, theanti-Siglec-9 antibody transiently activates and then inducesdegradation of Siglec-9. In some embodiments, the anti-Siglec-9 antibodytransiently activates and then induces cleavage of Siglec-9. In someembodiments, the anti-Siglec-9 antibody transiently activates and theninduces internalization of Siglec-9. In some embodiments, theanti-Siglec-9 antibody transiently activates and then induces sheddingof Siglec-9. In some embodiments, the anti-Siglec-9 antibody transientlyactivates and then induces downregulation of Siglec-9 expression. Insome embodiments, the anti-Siglec-9 antibody transiently activates andthen induces decreased expression of Siglec-9. In certain embodiments,the individual has a Siglec-9 variant allele.

As disclosed herein, agents of the present disclosure that bind orinteract with Siglec-9, such as anti-Siglec-9 antibodies of the presentdisclosure, may further be used for decreasing the activity,functionality, or survival of regulatory T cells, tumor-imbeddedimmunosuppressor dendritic cells, tumor-imbedded immunosuppressormacrophages, tumor-imbedded immunosuppressor neutrophils, tumor-imbeddedimmunosuppressor NK cells, myeloid derived suppressor cells,tumor-associated macrophages, tumor-associated neutrophils,tumor-associated NK cells, acute myeloid leukemia (AML) cells, chroniclymphocytic leukemia (CLL) cell, and/or chronic myeloid leukemia (CML)cells. In some embodiments, the present disclosure provides methods ofdecreasing the activity, functionality, or survival of regulatory Tcells, tumor-imbedded immunosuppressor dendritic cells, tumor-imbeddedimmunosuppressor macrophages, tumor-imbedded immunosuppressorneutrophils, tumor-imbedded immunosuppressor NK cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, tumor-associatedneutrophils, tumor-associated NK cells, acute myeloid leukemia (AML)cells, chronic lymphocytic leukemia (CLL) cell, or chronic myeloidleukemia (CML) cells in an individual in need thereof, by administeringto the individual a therapeutically effective amount of an agent thatbinds or interacts with Siglec-9. In some embodiments, the agent isselected from an antibody, an antagonist antibody, an inert antibody, anagonist antibody, a Siglec-9 ligand, a Siglec-9 ligand agonist fragment,a Siglec-9 immunoadhesin, a Siglec-9 ligand mimetic, a soluble Siglec-9receptor, a Siglec-9-Fc fusion protein, a soluble Siglec receptor thatbinds one or more Siglec-9 ligands, a Siglec-Fc fusion protein thatbinds one or more Siglec-9 ligands, and a small molecule compound. Insome embodiments, the agent is an isolated anti-Siglec-9 antibody oranti-Siglec-9 antibody conjugate of the present disclosure. In someembodiments, the anti-Siglec-9 antibody conjugate comprises ananti-Siglec-9 antibody conjugated to a detectable marker, a toxin, or atherapeutic agent.

As disclosed herein, agents of the present disclosure that bind orinteract with Siglec-9, such as anti-Siglec-9 antibodies of the presentdisclosure, may be used for decreasing cellular levels of Siglec-9 onone or more cells in vitro or in vivo, including without limitation, redblood cells, bacterial cells, apoptotic cells, nerve cells, glia cells,microglia, astrocytes, tumor cells, viruses, dendritic cells, Siglec-9ligands bound to beta amyloid plaques, Siglec-9 ligands bound to Tautangles, Siglec-9 ligands on disease-causing proteins, Siglec-9 ligandson disease-causing peptides, macrophages, neutrophils, natural killercells, monocytes, T cells, T helper cells, cytotoxic T cells, B cells,tumor-imbedded immunosuppressor dendritic cells, tumor-imbeddedimmunosuppressor macrophages, myeloid-derived suppressor cells, and/orregulatory T cells. In some embodiments, the present disclosure providesmethods of decreasing cellular levels of Siglec-9 on one or more cellsin an individual in need thereof, by administering to the individual atherapeutically effective amount of an agent that binds or interactswith Siglec-9. In some embodiments, the agent is selected from anantibody, an antagonist antibody, an inert antibody, an agonistantibody, a Siglec-9 ligand, a Siglec-9 ligand agonist fragment, aSiglec-9 immunoadhesin, a Siglec-9 ligand mimetic, a soluble Siglec-9receptor, a Siglec-9-Fc fusion protein, a soluble Siglec receptor thatbinds one or more Siglec-9 ligands, a Siglec-Fc fusion protein thatbinds one or more Siglec-9 ligands, and a small molecule compound. Insome embodiments, the agent is an isolated anti-Siglec-9 antibody oranti-Siglec-9 antibody conjugate of the present disclosure. In someembodiments, the anti-Siglec-9 antibody conjugate comprises ananti-Siglec-9 antibody conjugated to a detectable marker, a toxin, or atherapeutic agent. In some embodiments, the one or more cells areselected from red blood cells, bacterial cells, apoptotic cells, nervecells, glia cells, microglia, astrocytes, tumor cells, viruses,dendritic cells, Siglec-9 ligands bound to beta amyloid plaques,Siglec-9 ligands bound to Tau tangles, Siglec-9 ligands ondisease-causing proteins, Siglec-9 ligands on disease-causing peptides,macrophages, neutrophils, natural killer cells, monocytes, T cells, Thelper cells, cytotoxic T cells, B cells, tumor-imbeddedimmunosuppressor dendritic cells, tumor-imbedded immunosuppressormacrophages, myeloid-derived suppressor cells, regulatory T cells, andany combination thereof.

Cellular levels of Siglec-9 may refer to, without limitation, cellsurface levels of Siglec-9, intracellular levels of Siglec-9, and totallevels of Siglec-9. In some embodiments, a decrease in cellular levelsof Siglec-9 comprises decrease in cell surface levels of Siglec-9. Asused herein, an anti-Siglec-9 antibody decreases cell surface levels ofSiglec-9 if it induces a decrease of 21% or more in cell surface levelsof Siglec-9 as measured by any in vitro cell-based assays or suitable invivo model described herein or known in the art. In some embodiments, adecrease in cellular levels of Siglec-9 comprises a decrease inintracellular levels of Siglec-9. As used herein, an anti-Siglec-9antibody decreases intracellular levels of Siglec-9 if it induces adecrease of 21% or more in intracellular levels of Siglec-9 as measuredby any in vitro cell-based assays or suitable in vivo model describedherein or known in the art. In some embodiments, a decrease in cellularlevels of Siglec-9 comprises a decrease in total levels of Siglec-9. Asused herein, an anti-Siglec-9 antibody decreases total levels ofSiglec-9 if it induces a decrease of 21% or more in total levels ofSiglec-9 as measured by any in vitro cell-based assays or suitable invivo model described herein or known in the art. In some embodiments,the anti-Siglec-9 antibodies induce Siglec-9 degradation, Siglec-9cleavage, Siglec-9 internalization, Siglec-9 shedding, and/ordownregulation of Siglec-9 expression. In some embodiments, cellularlevels of Siglec-9 are measured on primary cells (e.g., dendritic cells,bone marrow-derived dendritic cells, monocytes, microglia, andmacrophages) or on cell lines utilizing an in vitro cell assay.

In some embodiments the individual has a heterozygous variant ofSiglec-9.

In some embodiments, the methods of the present disclosure may furtherinvolve the coadministration of Siglec-9 agents, such as anti-Siglec-9antibodies or bispecific anti-Siglec-9 antibodies, with antibodies thatbind to pattern recognition receptors, antibodies that bind to Toll-likereceptors, antibodies that bind to damage-associated molecular pattern(DAMP) receptors, and/or antibodies that bind to cytokine or antibodiesto interleukins).

In some embodiments, the methods of the present disclosure may furtherinclude administering to the individual at least one antibody thatspecifically binds to an inhibitory checkpoint molecule, and/or one ormore standard or investigational anti-cancer therapies. In someembodiments, the at least one antibody that specifically binds to aninhibitory checkpoint molecule is administered in combination with theanti-Siglec-9 antibody. In some embodiments, the at least one antibodythat specifically binds to an inhibitory checkpoint molecule is selectedfrom an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2antibody, an anti-PD-1 antibody, an anti-B7-H3 antibody, an anti-B7-H4antibody, and anti-HVEM antibody, an anti-B- and T-lymphocyte attenuator(BTLA) antibody, an anti-Killer inhibitory receptor (KIR) antibody, ananti-GAL9 antibody, an anti-TIM-1 antibody, an anti-TIM3 antibody, ananti-TIM-4 antibody, an anti-A2AR antibody, an anti-CD39 antibody, ananti-CD73 antibody, an anti-LAG-3 antibody, an anti-phosphatidylserineantibody, an anti-CD27 antibody, an anti-CD30 antibody, an anti-TNFaantibody, an anti-CD33 antibody, an anti-Siglec-5 antibody, ananti-Siglec-7 antibody, an anti-Siglec-11 antibody, an antagonisticanti-TREM1 antibody, an antagonistic anti-TREM2 antibody, an anti-TIGITantibody, an anti-VISTA antibody, an anti-CD2 antibody, an anti-CD5antibody, and any combination thereof. In some embodiments, the one ormore standard or investigational anti-cancer therapies are selected fromradiotherapy, cytotoxic chemotherapy, targeted therapy, imatinibtherapy, trastuzumab therapy, etanercept therapy, adoptive cell transfer(ACT) therapy, chimeric antigen receptor T cell transfer (CAR-T)therapy, vaccine therapy, and cytokine therapy.

In some embodiments, the methods of the present disclosure may furtherinclude administering to the individual at least one antibody thatspecifically binds to an inhibitory cytokine. In some embodiments, theat least one antibody that specifically binds to an inhibitory cytokineis administered in combination with the Siglec-9 agent, such as ananti-Siglec-9 antibody. In some embodiments, the at least one antibodythat specifically binds to an inhibitory cytokine is selected from ananti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, andany combination thereof.

In some embodiments, the methods of the present disclosure may furtherinclude administering to the individual at least one agonistic antibodythat specifically binds to a stimulatory checkpoint protein. In someembodiments, the at least one agonistic antibody that specifically bindsto a stimulatory checkpoint protein is administered in combination withthe Siglec-9 agent, such as an anti-Siglec-9 antibody. In someembodiments, the at least one agonistic antibody that specifically bindsto a stimulatory checkpoint protein is selected from an agonistanti-CD40 antibody, an agonist anti-OX40 antibody, an agonist anti-ICOSantibody, an agonist anti-CD28 antibody, an agonistic anti-TREM1antibody, an agonistic anti-TREM2 antibody, an agonist anti-CD137/4-1BBantibody, an agonist anti-CD27 antibody, an agonistanti-glucocorticoid-induced TNFR-related protein GITR antibody, anagonist anti-CD30 antibody, an agonist anti-BTLA antibody, an agonistanti-HVEM antibody, an agonist anti-CD2 antibody, an agonist anti-CD5antibody, and any combination thereof.

In some embodiments, the methods of the present disclosure may furtherinclude administering to the individual at least one stimulatorycytokine. In some embodiments, the at least one stimulatory cytokine isadministered in combination with the Siglec-9 agent, such as ananti-Siglec-9 antibody. In some embodiments, the at least onestimulatory cytokine is selected from FN-α4, IFN-β, IL-1β, TNF-α, IL-6,IL-8, CRP, IL-20 family members, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11,IL-12, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MTP-1-beta, and anycombination thereof.

Dementia

Dementia is a non-specific syndrome (i.e., a set of signs and symptoms)that presents as a serious loss of global cognitive ability in apreviously unimpaired person, beyond what might be expected from normalageing. Dementia may be static as the result of a unique global braininjury. Alternatively, dementia may be progressive, resulting inlong-term decline due to damage or disease in the body. While dementiais much more common in the geriatric population, it can also occurbefore the age of 65. Cognitive areas affected by dementia include,without limitation, memory, attention span, language, and problemsolving. Generally, symptoms must be present for at least six months tobefore an individual is diagnosed with dementia.

Exemplary forms of dementia include, without limitation, frontotemporaldementia, Alzheimer's disease, vascular dementia, semantic dementia, anddementia with Lewy bodies.

In some embodiments, administering a Siglec-9 agent of the presentdisclosure, such as an anti-Siglec-9 antibody of the present disclosure,can prevent, reduce the risk, and/or treat dementia. In someembodiments, administering a Siglec-9 agent, such as an anti-Siglec-9antibody, may modulate one or more Siglec-9 activities in an individualhaving dementia.

Frontotemporal Dementia

Frontotemporal dementia (FTD) is a condition resulting from theprogressive deterioration of the frontal lobe of the brain. Over time,the degeneration may advance to the temporal lobe. Second only toAlzheimer's disease (AD) in prevalence, FTD accounts for 20% ofpre-senile dementia cases. The clinical features of FTD include memorydeficits, behavioral abnormalities, personality changes, and languageimpairments (Cruts, M. & Van Broeckhoven, C., Trends Genet. 24:186-194(2008); Neary, D., et al., Neurology 51:1546-1554 (1998); Ratnavalli,E., Brayne, C., Dawson, K. & Hodges, J. R., Neurology 58:1615-1621(2002)).

A substantial portion of FTD cases are inherited in an autosomaldominant fashion, but even in one family, symptoms can span a spectrumfrom FTD with behavioral disturbances, to Primary Progressive Aphasia,to Cortico-Basal Ganglionic Degeneration. FTD, like mostneurodegenerative diseases, can be characterized by the pathologicalpresence of specific protein aggregates in the diseased brain.Historically, the first descriptions of FTD recognized the presence ofintraneuronal accumulations of hyperphosphorylated Tau protein inneurofibrillary tangles or Pick bodies. A causal role for themicrotubule associated protein Tau was supported by the identificationof mutations in the gene encoding the Tau protein in several families(Hutton, M., et al., Nature 393:702-705 (1998). However, the majority ofFTD brains show no accumulation of hyperphosphorylated Tau but doexhibit immunoreactivity to ubiquitin (Ub) and TAR DNA binding protein(TDP43) (Neumann, M., et al., Arch. Neurol. 64:1388-1394 (2007)). Amajority of those FTD cases with Ub inclusions (FTD-U) were shown tocarry mutations in the Progranulin gene.

In some embodiments, administering a Siglec-9 agent of the presentdisclosure, such as an anti-Siglec-9 antibody of the present disclosure,can prevent, reduce the risk, and/or treat FTD. In some embodiments,administering a Siglec-9 agent, such as an anti-Siglec-9 antibody, maymodulate one or more Siglec-9 activities in an individual having FTD.

Alzheimer's Disease

Alzheimer's disease (AD), is the most common form of dementia. There isno cure for the disease, which worsens as it progresses, and eventuallyleads to death. Most often, AD is diagnosed in people over 65 years ofage. However, the less-prevalent early-onset Alzheimer's can occur muchearlier.

Common symptoms of Alzheimer's disease include, behavioral symptoms,such as difficulty in remembering recent events; cognitive symptoms,confusion, irritability and aggression, mood swings, trouble withlanguage, and long-term memory loss. As the disease progresses bodilyfunctions are lost, ultimately leading to death. Alzheimer's diseasedevelops for an unknown and variable amount of time before becomingfully apparent, and it can progress undiagnosed for years.

In some embodiments, administering a Siglec-9 agent of the presentdisclosure, such as an anti-Siglec-9 antibody of the present disclosure,can prevent, reduce the risk, and/or treat Alzheimer's disease. In someembodiments, administering a Siglec-9 agent, such as an anti-Siglec-9antibody, may modulate one or more Siglec-9 activities in an individualhaving Alzheimer's disease.

Parkinson's Disease

Parkinson's disease, which may be referred to as idiopathic or primaryparkinsonism, hypokinetic rigid syndrome (HRS), or paralysis agitans, isa neurodegenerative brain disorder that affects motor system control.The progressive death of dopamine-producing cells in the brain leads tothe major symptoms of Parkinson's. Most often, Parkinson's disease isdiagnosed in people over 50 years of age. Parkinson's disease isidiopathic (having no known cause) in most people. However, geneticfactors also play a role in the disease.

Symptoms of Parkinson's disease include, without limitation, tremors ofthe hands, arms, legs, jaw, and face, muscle rigidity in the limbs andtrunk, slowness of movement (bradykinesia), postural instability,difficulty walking, neuropsychiatric problems, changes in speech orbehavior, depression, anxiety, pain, psychosis, dementia,hallucinations, and sleep problems.

In some embodiments, administering a Siglec-9 agent of the presentdisclosure, such as an anti-Siglec-9 antibody of the present disclosure,can prevent, reduce the risk, and/or treat Parkinson's disease. In someembodiments, administering a Siglec-9 agent, such as an anti-Siglec-9antibody, may modulate one or more Siglec-9 activities in an individualhaving Parkinson's disease.

Amyotrophic Lateral Sclerosis (ALS)

As used herein, amyotrophic lateral sclerosis (ALS) or, motor neurondisease or, Lou Gehrig's disease are used interchangeably and refer to adebilitating disease with varied etiology characterized by rapidlyprogressive weakness, muscle atrophy and fasciculations, musclespasticity, difficulty speaking (dysarthria), difficulty swallowing(dysphagia), and difficulty breathing (dyspnea).

It has been shown that Progranulin plays a role in ALS (Schymick, J C etal., (2007) J Neurol Neurosurg Psychiatry.; 78:754-6) and protects againthe damage caused by ALS causing proteins such as TDP-43 (Laird, A S etal., (2010). PLoS ONE 5: e13368). It was also demonstrated that pro-NGFinduces p75 mediated death of oligodendrocytes and corticospinal neuronsfollowing spinal cord injury (Beatty et al., Neuron (2002), 36, pp.375-386; Gichl et al, Proc. Natl. Acad. Sci USA (2004), 101, pp6226-30).

In some embodiments, administering a Siglec-9 agent of the presentdisclosure, such as an anti-Siglec-9 antibody of the present disclosure,can prevent, reduce the risk, and/or treat ALS. In some embodiments,administering a Siglec-9 agent, such as an anti-Siglec-9 antibody, maymodulate one or more Siglec-9 activities in an individual havingamyotrophic lateral sclerosis.

Huntington's Disease

Huntington's disease (HD) is an inherited neurodegenerative diseasecaused by an autosomal dominant mutation in the Huntingtin gene (HTT).Expansion of a cytokine-adenine-guanine (CAG) triplet repeat within theHuntingtin gene results in production of a mutant form of the Huntingtinprotein (Htt) encoded by the gene. This mutant Huntingtin protein (mHtt)is toxic and contributes to neuronal death. Symptoms of Huntington'sdisease most commonly appear between the ages of 35 and 44, althoughthey can appear at any age.

Symptoms of Huntington's disease, include, without limitation, motorcontrol problems, jerky, random movements (chorea), abnormal eyemovements, impaired balance, seizures, difficulty chewing, difficultyswallowing, cognitive problems, altered speech, memory deficits,thinking difficulties, insomnia, fatigue, dementia, changes inpersonality, depression, anxiety, and compulsive behavior.

In some embodiments, administering a Siglec-9 agent of the presentdisclosure, such as an anti-Siglec-9 antibody of the present disclosure,can prevent, reduce the risk, and/or treat Huntington's disease (HD). Insome embodiments, administering a Siglec-9 agent, such as ananti-Siglec-9 antibody, may modulate one or more Siglec-9 activities inan individual having Huntington's disease.

Taupathy Disease

Taupathy diseases, or Tauopathies, are a class of neurodegenerativedisease caused by aggregation of the microtubule-associated protein tauwithin the brain. Alzheimer's disease (AD) is the most well-knowntaupathy disease, and involves an accumulation of tau protein withinneurons in the form of insoluble neurofibrillary tangles (NFTs). Othertaupathy diseases and disorders include progressive supranuclear palsy,dementia pugilistica (chromic traumatic encephalopathy), frontotemporaldementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease(Parkinson-dementia complex of Guam), Tangle-predominant dementia,Ganglioglioma and gangliocytoma, Meningioangiomatosis, Subacutesclerosing panencephalitis, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasaldegeneration, Argyrophilic grain disease (AGD), Huntington's disease,and frontotemporal lobar degeneration.

In some embodiments, administering a Siglec-9 agent of the presentdisclosure, such as an anti-Siglec-9 antibody of the present disclosure,can prevent, reduce the risk, and/or treat taupathy disease. In someembodiments, administering a Siglec-9 agent, such as an anti-Siglec-9antibody, may modulate one or more Siglec-9 activities in an individualhaving a taupathy disease.

Multiple Sclerosis

Multiple sclerosis (MS) can also be referred to as disseminatedsclerosis or encephalomyelitis disseminata. MS is an inflammatorydisease in which the fatty myelin sheaths around the axons of the brainand spinal cord are damaged, leading to demyelination and scarring aswell as a broad spectrum of signs and symptoms. MS affects the abilityof nerve cells in the brain and spinal cord to communicate with eachother effectively. Nerve cells communicate by sending electrical signalscalled action potentials down long fibers called axons, which arecontained within an insulating substance called myelin. In MS, thebody's own immune system attacks and damages the myelin. When myelin islost, the axons can no longer effectively conduct signals. MS onsetusually occurs in young adults, and is more common in women.

Symptoms of MS include, without limitation, changes in sensation, suchas loss of sensitivity or tingling; pricking or numbness, such ashypoesthesia and paresthesia; muscle weakness; clonus; muscle spasms;difficulty in moving; difficulties with coordination and balance, suchas ataxia; problems in speech, such as dysarthria, or in swallowing,such as dysphagia; visual problems, such as nystagmus, optic neuritisincluding phosphenes, and diplopia; fatigue; acute or chronic pain; andbladder and bowel difficulties; cognitive impairment of varying degrees;emotional symptoms of depression or unstable mood; Uhthoffs phenomenon,which is an exacerbation of extant symptoms due to an exposure to higherthan usual ambient temperatures; and Lhernitte's sign, which is anelectrical sensation that runs down the back when bending the neck.

In some embodiments, administering a Siglec-9 agent of the presentdisclosure, such as an anti-Siglec-9 antibody of the present disclosure,can prevent, reduce the risk, and/or treat multiple sclerosis. In someembodiments, administering a Siglec-9 agent, such as an anti-Siglec-9antibody, may modulate one or more Siglec-9 activities in an individualhaving multiple sclerosis.

Cancer

Further aspects of the present disclosure provide methods forpreventing, reducing risk, or treating cancer, by administering to anindividual in need thereof a therapeutically effective amount of aSiglec-9 agent of the present disclosure, such as an isolatedanti-Siglec-9 antibody of the present disclosure. Any of the isolatedantibodies of the present disclosure may be used in these methods. Insome embodiments, the isolated antibody is an agonist antibody of thepresent disclosure. In other embodiments, the isolated antibody is anantagonist antibody of the present disclosure. In other embodiments, theisolated antibody is an inert antibody of the present disclosure. Inother embodiments, the isolated antibody is an antibody conjugate of thepresent disclosure.

As disclosed herein, the tumor microenvironment is known to contain aheterogeneous immune infiltrate, which includes T lymphocytes,macrophages, neutrophils, NK cells, and cells of myeloid/granulocyticlineage. The presence and activity of T-regulatory cells, tumor-imbeddedimmunosuppressor myeloid cells, and/or M2-macrophages, M2-neutrophils,and/or M2-NK cells in tumors is associated with poor prognosis. Incontrast, the presence and activity of cytotoxic T cells is beneficialfor cancer therapy. Therapies that directly or indirectly enhance theactivity of cytotoxic T cells and reduce the number and activity of thevarious immunosuppressor cells, are expected to provide significanttherapeutic benefit. A seminal preclinical study has shown synergiesbetween drugs that target immunosuppressor cells (e.g., CSF1/CSF1Rblocking antibodies) and immune checkpoint blocking antibodies thatactivate cytotoxic T cells, indicating that manipulating both cell typesshows efficacy in tumor models where individual therapies are poorlyeffective (Zhu Y; Cancer Res. 2014 Sep. 15; 74(18):5057-69). Therefore,in some embodiments, blocking Siglec-9, which is expressed on myeloidcells, subset of T cells, and tumor-associated immune cells, maystimulate beneficial anti-tumor immune response, resulting in atherapeutic anti-tumor immune response.

In some embodiments, the methods for preventing, reducing risk, ortreating an individual having cancer further include administering tothe individual at least one antibody that specifically binds to aninhibitory checkpoint molecule. Examples of antibodies that specificallybind to an inhibitory checkpoint molecule include, without limitation,an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody,an anti-PD-1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody,and anti-HVEM antibody, an anti-B- and T-lymphocyte attenuator (BTLA)antibody, an anti-Killer inhibitory receptor (KIR) antibody, ananti-GAL9 antibody, an anti-TIM-1 antibody, an anti-TIM3 antibody, ananti-TIM-4 antibody, an anti-A2AR antibody, an anti-CD39 antibody, ananti-CD73 antibody, an anti-LAG-3 antibody, an anti-phosphatidylserineantibody, an anti-CD27 antibody, an anti-CD30 antibody, an anti-TNFaantibody, an anti-CD33 antibody, an anti-Siglec-5 antibody, ananti-Siglec-7 antibody, an anti-Siglec-11 antibody, an antagonisticanti-TREM1 antibody, an antagonistic anti-TREM2 antibody, an anti-TIGITantibody, an anti-VISTA antibody, an anti-CD2 antibody, an anti-CD5antibody, and any combination thereof. In some embodiments, the at leastone antibody that specifically binds to an inhibitory checkpointmolecule is administered in combination with a Siglec-9 agent of thepresent disclosure, such as an antagonist anti-Siglec-9 antibody of thepresent disclosure.

In some embodiments, a cancer to be prevented or treated by the methodsof the present disclosure includes, without limitation, squamous cellcancer (e.g., epithelial squamous cell cancer), lung cancer includingsmall-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung and squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer and gastrointestinal stromal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, cancer of the urinary tract, hepatoma, breast cancer,colon cancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, melanoma, superficial spreading melanoma,lentigo maligna melanoma, acral lentiginous melanomas, nodularmelanomas, multiple myeloma and B cell lymphoma; chronic lymphocyticleukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia;chronic myeloblastic leukemia; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),Meigs' syndrome, brain, as well as head and neck cancer, and associatedmetastases. In some embodiments, the cancer is colorectal cancer. Insome embodiments, the cancer is selected from non-small cell lungcancer, glioblastoma, neuroblastoma, renal cell carcinoma, bladdercancer, ovarian cancer, melanoma, breast carcinoma, gastric cancer, andhepatocellular carcinoma. In some embodiments, the cancer istriple-negative breast carcinoma. In some embodiments, the cancer may bean early stage cancer or a late stage cancer. In some embodiments, thecancer may be a primary tumor. In some embodiments, the cancer may be ametastatic tumor at a second site derived from any of the above types ofcancer.

In some embodiments, Siglec-9 agents of the present disclosure, such asanti-Siglec-9 antibodies of the present disclosure, may be used forpreventing, reducing risk, or treating cancer, including, withoutlimitation, bladder cancer breast cancer, colon and rectal cancer,endometrial cancer, kidney cancer, renal cell cancer, renal pelviscancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer.

In some embodiments, the present disclosure provides methods ofpreventing, reducing risk, or treating an individual having cancer, byadministering to the individual a therapeutically effective amount of aSiglec-9 agent of the present disclosure, such as an anti-Siglec-9antibody of the present disclosure.

In some embodiments, the method further includes administering to theindividual at least one antibody that specifically binds to aninhibitory immune checkpoint molecule, and/or another standard orinvestigational anti-cancer therapy. In some embodiments, the at leastone antibody that specifically binds to an inhibitory checkpointmolecule is administered in combination with the Siglec-9 agent, such asan anti-Siglec-9 antibody of the present disclosure. In someembodiments, the at least one antibody that specifically binds to aninhibitory checkpoint molecule is selected from an anti-PD-L1 antibody,an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1 antibody,an anti-B7-H3 antibody, an anti-B7-H4 antibody, and anti-HVEM antibody,an anti-B- and T-lymphocyte attenuator (BTLA) antibody, an anti-Killerinhibitory receptor (KIR) antibody, an anti-GAL9 antibody, an anti-TIM-1antibody, an anti-TIM3 antibody, an anti-TIM-4 antibody, an anti-A2ARantibody, an anti-CD39 antibody, an anti-CD73 antibody, an anti-LAG-3antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, ananti-CD30 antibody, an anti-TNFa antibody, an anti-CD33 antibody, ananti-Siglec-5 antibody, an anti-Siglec-7 antibody, an anti-Siglec-11antibody, an antagonistic anti-TREM1 antibody, an antagonisticanti-TREM2 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, ananti-CD2 antibody, an anti-CD5 antibody, and any combination thereof. Insome embodiments, the standard or investigational anti-cancer therapy isone or more therapies selected from radiotherapy, cytotoxicchemotherapy, targeted therapy, imatinib (Gleevec®), trastuzumab(Herceptin®), adoptive cell transfer (ACT), chimeric antigen receptor Tcell transfer (CAR-T), vaccine therapy, and cytokine therapy.

In some embodiments, the method further includes administering to theindividual at least one antibody that specifically binds to aninhibitory cytokine. In some embodiments, the at least one antibody thatspecifically binds to an inhibitory cytokine is administered incombination with the Siglec-9 agent, such as an anti-Siglec-9 antibodyof the present disclosure. In some embodiments, the at least oneantibody that specifically binds to an inhibitory cytokine is selectedfrom an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2antibody, and any combination thereof.

In some embodiments, the method further includes administering to theindividual at least one agonistic antibody that specifically binds to astimulatory immune checkpoint protein. In some embodiments, the at leastone agonistic antibody that specifically binds to a stimulatorycheckpoint protein is administered in combination with the Siglec-9agent, such as an anti-Siglec-9 antibody of the present disclosure. Insome embodiments, the at least one agonistic antibody that specificallybinds to a stimulatory checkpoint protein is selected from an agonistanti-CD40 antibody, an agonist anti-OX40 antibody, an agonist anti-ICOSantibody, an agonist anti-CD28 antibody, an agonistic anti-TREM1antibody, an agonistic anti-TREM2 antibody, an agonist anti-CD137/4-1BBantibody, an agonist anti-CD27 antibody, an agonistanti-glucocorticoid-induced TNFR-related protein GITR antibody, anagonist anti-CD30 antibody, an agonist anti-BTLA antibody, an agonistanti-HVEM antibody, an agonist anti-CD2 antibody, an agonist anti-CD5antibody, and any combination thereof.

In some embodiments, the method further includes administering to theindividual at least one stimulatory cytokine. In some embodiments, theat least one stimulatory cytokine is administered in combination withthe Siglec-9 agent, such as an anti-Siglec-9 antibody of the presentdisclosure. In some embodiments, the at least one stimulatory cytokineis selected from FN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, IL-20family members, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-17,IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1-beta, and any combinationthereof.

Kits/Articles of Manufacture

The present disclosure also provides kits and/or articles of manufacturecontaining a Siglec-9 agent of the present disclosure (e.g., ananti-Siglec-9 antibody described herein), or a functional fragmentthereof. Kits and/or articles of manufacture of the present disclosuremay include one or more containers comprising a purified antibody of thepresent disclosure. In some embodiments, the kits and/or articles ofmanufacture further include instructions for use in accordance with themethods of this disclosure. In some embodiments, these instructionscomprise a description of administration of the Siglec-9 agent of thepresent disclosure (e.g., an anti-Siglec-9 antibody described herein) toprevent, reduce risk, or treat an individual having a disease, disorder,or injury selected from dementia, frontotemporal dementia, Alzheimer'sdisease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease,normal pressure hydrocephalus, amyotrophic lateral sclerosis,Huntington's disease, taupathy disease, Nasu-Hakola disease, stroke,acute trauma, chronic trauma, lupus, acute and chronic colitis,rheumatoid arthritis, wound healing, Crohn's disease, inflammatory boweldisease, ulcerative colitis, obesity, malaria, essential tremor, centralnervous system lupus, Behcet's disease, Parkinson's disease, dementiawith Lewy bodies, multiple system atrophy, Shy-Drager syndrome,progressive supranuclear palsy, cortical basal ganglionic degeneration,acute disseminated encephalomyelitis, granulomartous disorders,sarcoidosis, diseases of aging, seizures, spinal cord injury, traumaticbrain injury, age related macular degeneration, glaucoma, retinitispigmentosa, retinal degeneration, respiratory tract infection, sepsis,eye infection, systemic infection, lupus, arthritis, multiple sclerosis,low bone density, osteoporosis, osteogenesis, osteopetrotic disease,Paget's disease of bone, and cancer including bladder cancer, braincancer, breast cancer, colon cancer, rectal cancer, endometrial cancer,kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lungcancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostatecancer, ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),chronic myeloid leukemia (CML), multiple myeloma, polycythemia vera,essential thrombocytosis, primary or idiopathic myelofibrosis, primaryor idiopathic myelosclerosis, myeloid-derived tumors, tumors thatexpress Siglec-9, thyroid cancer, infections, CNS herpes, parasiticinfections, Trypanosome infection, Cruzi infection, Pseudomonasaeruginosa infection, Leishmania donovani infection, group BStreptococcus infection, Campylobacter jejuni infection, Neisseriameningiditis infection, type I HIV, and Haemophilus influenza, accordingto any methods of this disclosure.

In some embodiments, the instructions comprise a description of how todetect a Siglec-9 protein, for example in an individual, in a tissuesample, or in a cell. The kit and/or article of manufacture may furthercomprise a description of selecting an individual suitable for treatmentbased on identifying whether that individual has the disease and thestage of the disease.

In some embodiments, the kits and/or articles of manufacture may furtherinclude another antibody of the present disclosure (e.g., at least oneantibody that specifically binds to an inhibitory checkpoint molecule,at least one antibody that specifically binds to an inhibitory cytokine,and/or at least one agonistic antibody that specifically binds to astimulatory checkpoint protein) and/or at least one stimulatorycytokine. In some embodiments, the kits and/or articles of manufacturemay further include instructions for using the antibody and/orstimulatory cytokine in combination with a Siglec-9 agent of the presentdisclosure (e.g., an anti-Siglec-9 antibody described herein),instructions for using a Siglec-9 agent of the present disclosure (e.g.,an anti-Siglec-9 antibody described herein) in combination with anantibody and/or stimulatory cytokine, or instructions for using aSiglec-9 agent of the present disclosure (e.g., an anti-Siglec-9antibody described herein) and an antibody and/or stimulatory cytokine,according to any methods of this disclosure.

The instructions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits and/or articles ofmanufacture of the present disclosure are typically written instructionson a label or package insert (e.g., a paper sheet included in the kit),but machine-readable instructions (e.g., instructions carried on amagnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, e.g., a disease of the present disclosure. Instructions may beprovided for practicing any of the methods described herein.

The kits and/or articles of manufacture of this disclosure are insuitable packaging. Suitable packaging includes, but is not limited to,vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plasticbags), and the like. Also contemplated are packages for use incombination with a specific device, such as an inhaler, nasaladministration device (e.g., an atomizer) or an infusion device such asa minipump. A kit and/or article of manufacture may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The container may also have a sterile access port (e.g., thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is a Siglec-9 agent of the present disclosure (e.g.,an anti-Siglec-9 antibody described herein). The container may furthercomprise a second pharmaceutically active agent.

Kits and/or articles of manufacture may optionally provide additionalcomponents such as buffers and interpretive information. Normally, thekit comprises a container and a label or package insert(s) on orassociated with the container.

Diagnostic Uses

The Siglec-9 agents of the present disclosure, such as the isolatedantibodies of the present disclosure (e.g., an anti-Siglec-9 antibodydescribed herein) also have diagnostic utility. This disclosuretherefore provides for methods of using the antibodies of thisdisclosure, or functional fragments thereof, for diagnostic purposes,such as the detection of a Siglec-9 protein in an individual or intissue samples derived from an individual.

In some embodiments, the individual is a human. In some embodiments, theindividual is a human patient suffering from, or at risk for developinga disease, disorder, or injury of the present disclosure. In someembodiments, the diagnostic methods involve detecting a Siglec-9 proteinin a biological sample, such as a biopsy specimen, a tissue, or a cell.A Siglec-9 agent of the present disclosure (e.g., an anti-Siglec-9antibody described herein) is contacted with the biological sample andantigen-bound antibody is detected. For example, a biopsy specimen maybe stained with an anti-Siglec-9 antibody described herein in order todetect and/or quantify disease-associated cells. The detection methodmay involve quantification of the antigen-bound antibody. Antibodydetection in biological samples may occur with any method known in theart, including immunofluorescence microscopy, immunocytochemistry,immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, ormicro-positron emission tomography. In certain embodiments, the antibodyis radiolabeled, for example with ¹⁸F and subsequently detectedutilizing micro-positron emission tomography analysis. Antibody-bindingmay also be quantified in a patient by non-invasive techniques such aspositron emission tomography (PET), X-ray computed tomography,single-photon emission computed tomography (SPECT), computed tomography(CT), and computed axial tomography (CAT).

In other embodiments, an isolated antibody of the present disclosure(e.g., an anti-Siglec-9 antibody described herein) may be used to detectand/or quantify, for example, microglia in a brain specimen taken from apreclinical disease model (e.g., a non-human disease model). As such, anisolated antibody of the present disclosure (e.g., an anti-Siglec-9antibody described herein) may be useful in evaluating therapeuticresponse after treatment in a model for a nervous system disease orinjury such as frontotemporal dementia, Alzheimer's disease, vasculardementia, seizures, retinal dystrophy, atherosclerotic vasculardiseases, Nasu-Hakola disease, or multiple sclerosis, as compared to acontrol.

The present disclosure will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingthe scope of the present disclosure. All citations throughout thedisclosure are hereby expressly incorporated by reference.

EXAMPLES Example 1: Production, Identification, and Characterization ofAnti-Siglec-9 Antibodies

Introduction

The amino acid sequence of human Siglec-9 is set forth below in SEQ IDNO: 1. Human Siglec-9 contains a signal sequence located at amino acidresidues 1-17 SEQ ID NO: 1, an extracellular immunoglobulin-likevariable-type (IgV) domain located at amino acid residues 20-140 of SEQID NO: 1, two Ig-like C2-type domains located at amino acid residues146-229 and 236-336 of SEQ ID NO: 1, a transmembrane domain located atamino acid residues 348-370 of SEQ ID NO: 1, an ITIM motif 1 located atamino acid residues 431-436 of SEQ ID NO: 1, and a SLAM-like motiflocated at amino acid residues 454-459 of SEQ ID NO: 1. An alignment ofhuman Siglec-9 with Siglec-9 homologs is shown in FIG. 1A.

Siglec-9 amino acid sequence (SEQ ID NO: 1):

        10         20         30         40         50MLLLLLPLLW GRERAEGQTS KLLTMQSSVT VQEGLCVHVP CSFSYPSHGW        60         70         80         90        100IYPGPVVHGY WFREGANTDQ DAPVATNNPA RAVWEETRDR FHLLGDPHTK       110        120        130        140        150NCTLSIRDAR RSDAGRYFFR MEKGSIKWNY KHHRLSVNVT ALTHRPNILI       160        170        180        190        200PGTLESGCPQ NLTCSVPWAC EQGTPPMISW IGTSVSPLDP STTRSSVLTL       210        220        230        240        250IPQPQDHGTS LTCQVTFPGA SVTTNKTVHL NVSYPPQNLT MTVFQGDGTV       260        270        280        290        300STVLGNGSSL SLPEGQSLRL VCAVDAVDSN PPARLSLSWR GLTLGPSQPS       310        320        330        340        350NPGVLELPWV HLRDAAEFTC RAQNPLGSQQ VYLNVSLQSK ATSGVTQGVV       360        370        380        390        400GGAGATALVF LSFCVIFVVV RSCRKKSARP AAGVGDTGIE DANAVRGSAS       410        420        430        440        450QGPLTEPWAE DSPPDQPPPA SARSSVGEGE LQYASLSFQM VKPWDSRGQE        460ATDTEYSEIK IHR

The purpose of the following Example was to produce Siglec-9-bindingantibodies (e.g., antagonistic antibodies, Siglec-9 specific antibodies)that enhance the beneficial effects of dendritic cells, monocytes,macrophages, neutrophils, NK cells, T cells, and/or microglia.Antibodies that bind the extracellular domain of Siglec-9, particularlythe IgV domain (amino acid residues 20-140 of SEQ ID NO: 1) aregenerated using mouse hybridoma technology, phage display technology,and yeast display technology. Antibodies are identified and thenscreened for their ability to compete with Siglec-9 ligands on bindingto Siglec-9, to induce Siglec-9 downregulation, to induce Siglec-9desensitization, to induce Siglec-9 degradation, to induce Siglec-9targeting to the lysosome, to induce Siglec-9 cleavage, to modulateSiglec-9 signaling and/or one or more functions in cells and in animalsin vivo, as described in the following Examples. Exemplary ligands boundby Siglec-9 are depicted in FIG. 2 and FIG. 3.

For example, anti-Siglec-9 antibodies are selected that target the IgVdomain (amino acid residues 20-140) of Siglec-9. The IgV domain binds tosialic acid targets, and this binding can be blocked with the antibody.Thus, amino acid residues 20-140 correspond to a Siglec-9 peptide targetfor antibodies that will block binding of Siglec-9 to one or moreendogenous targets (e.g., ligands).

Another approach for identifying a useful site within human Siglec-9protein is by selecting antibodies targeting sites that are not presenton Siglec-9m as compared to Siglec-9M, which are generally found withinthe IgV domain. Siglec-9m is not able to inhibit clearance of theamyloid beta peptide. Another approach for identifying useful antibodiesis to select for antibodies that decrease the level of Siglec-9 on thecell surface of monocytes, macrophages, neutrophils, NK cells, dendriticcells, microglia, and/or T cells.

As described herein, nine anti-Siglec-9 antibodies were identified andcharacterized.

Results

Anti-Siglec-9 Antibody Production

Immunization Procedure

Rapid Prime Method:

Four 50-day old female BALB/c mice were immunized using the followingprocedure. A series of subcutaneous aqueous injections containing humanSiglec-9 antigen but no adjuvant was given over a period of 19 days.Mice were housed in a ventilated rack system from Lab Products. All fourmice were euthanized on Day 19 and lymphocytes were harvested forhybridoma cell line generation.

Standard Method:

Four 50-day old female BALB/c or NZB/W mice were immunized using thefollowing procedure. Mice were housed in a ventilated rack system fromLab Products. Mice were injected intraperitoneally every 3 weeks with ahuman Siglec-9 antigen mixed in CpG-ODN adjuvant at 25 μg proteinantigen per mouse (total volume 125 μL per mouse). Test bleeds were doneby saphenous vein lancing seven days after the second boost. The testbleed (immune sera) was tested by indirect ELISA assay to determine thebest two responding mice for the fusion. The mice may require a 3rd and4th boost and another test bleed 7 days after boost to assess titrebefore fusion. When the antibody titre is high enough the best tworesponding mice are given a final intravenous boost via lateral tailvein. Four days after the IV boost the mice were euthanized for fusion.The spleens were harvested and lymphocytes isolated from the spleen wereused in the fusion process to produce hybridomas.

Hybridoma Development

Lymphocytes were isolated and fused with murine SP2/0 myeloma cells inthe presence of poly-ethylene glycol (PEG 1500) as per standard RocheProtocol. Fused cells were cultured using a single-step cloning method(HAT selection). This method uses a semi-solid methylcellulose-based HATselective medium to combine the hybridoma selection and cloning into onestep. Single cell-derived hybridomas grow to form monoclonal colonies onthe semi-solid media. Ten days after the fusion event, 948 of theresulting hybridoma clones were transferred to 96-well tissue cultureplates and grown in HT containing medium until mid-log growth wasreached (5 days).

Hybridoma Screening

Tissue culture supernatants from the 948 hybridomas were tested byindirect ELISA on screening antigen (Primary Screening) and probed forboth IgG and IgM antibodies using a Goat anti-IgG/IgM(H&L)-HRP secondaryand developed with TMB substrate. Clones >0.2 OD in this assay weretaken to the next round of testing. Positive cultures were retested onscreening antigen to confirm secretion and on an irrelevant antigen(Human Transferrin) to eliminate non-specific or “sticky” mAbs and ruleout false positives. All clones of interest were isotyped by antibodytrapping ELISA to determine if they are IgG or IgM isotype.

Hybridoma Cell Culture

The hybridoma cell lines of interest were maintained in culture in24-well culture plates for 32 days post transfer to 96-well plates. Thisis referred to as the stability period and tests whether clones remainstable and secreting. During this stability period time temporary frozencell line back up is made of all the clones of interest for −80° C.storage (viable 6 months). Hybridomas were periodically tested duringthis time period for secretion and specificity.

Subcloning

The top hybridoma cell lines (clones) were subcloned to ensuremonoclonality. Subcloning was performed by plating parental clones outagain using the single-step cloning system. Between 24 and 90 subcloneswere transferred to 96-well culture plates. Subclones were screened byindirect ELISA and antibody trapping ELISA. The top subclones for eachparent were taken for expansion in culture. Any parental clones thatwere <50% clonal had a second round of subcloning performed.

The antibodies were then screened for Siglec-9 binding. Antibodies thatwere positive for binding to human Siglec-9 were tested for ability toblock ligand binding and ability to reduce surface levels of Siglec-9 inmultiple cell types. Six Siglec-9 antibodies were identified. The binand isotype category are listed in Table 1.

TABLE 1 Anti-Siglec-9 antibodies Ab ID Bin Ab Isotype 2D4 1 mIgG1 2D5 5mIgG1 5B1 4 mIgG1 6B2 3 mIgG1/2b* 6D8 3 mIgG2b 7H12 5 mIgG1/2a*

Antibody Heavy Chain and Light Chain Variable Domain Sequences

Using standard techniques, the amino acid sequences encoding the lightchain variable and the heavy chain variable domains of the generatedantibodies were determined. The EU or Kabat light chain HVR sequences ofthe antibodies are set forth in Table 2. The EU or Kabat heavy chain HVRsequences of the antibodies are set forth in Table 3. The EU or Kabatlight chain framework (FR) sequences of the antibodies are set forth inTable 4A. The EU or Kabat heavy chain framework (FR) sequences of theantibodies are set forth in Table 4B.

TABLE 2EU or Kabat light chain HVR sequences of anti-Siglec-9 antibodies AbHVR L1 HVR L2 HVR L3 2D4 RASQEISGYLG STSTLDS (SEQ ID NO: 10)LQYASYPPT (SEQ ID NO: 14) (SEQ ID NO: 6) 2D5 KSSQSLLDSDGKTYLNLVCKLDS (SEQ ID NO: 11) WQGTHFPQT (SEQ ID NO: 15) (SEQ ID NO: 7) 5B1KSSQSLLYTNGKTYLN LVSKLES (SEQ ID NO: 12) LQATHIPLT (SEQ ID NO: 16)(SEQ ID NO: 8) 6D8 HTTRGIYWYKG CARNGEG (SEQ ID NO: 13)AQFYQFPWT (SEQ ID NO: 17) (SEQ ID NO: 9) 7H12 KSSQSLLYTNGKTYLNLVSKLES (SEQ ID NO: 12) LQAPHFPLT (SEQ ID NO: 18) (SEQ ID NO: 8) 5C6SSSQSLVHSNGNTYLH KVSNRFS (SEQ ID NO: 174) SQNTHVPLT (SEQ ID NO: 172)(SEQ ID NO: 176) 12B12 RASENVDSYGISFMH RASNLES (SEQ ID NO: 175)QQSNEDPWT (SEQ ID NO: 173) (SEQ ID NO: 177) 17C2 SSSQSLVHSNGNTYLHKVSNRFS (SEQ ID NO: 174) SQNTHVPLT (SEQ ID NO: 172) (SEQ ID NO: 176)

TABLE 3EU or Kabat heavy chain HVR sequences of anti-Siglec-9 antibodies AbHVR H1 HVR H2 HVR H3 2D4 FTFSNYAMS (SEQ ID NO: 19) VATINNGGSYTYYSVRRDYGTSDFDY (SEQ ID NO: 22) (SEQ ID NO: 26) 2D5 YAFSSYWMN (SEQ IDIGRIY PRDGDTN YN ARWLERFAY NO: 20) (SEQ ID NO: 23) (SEQ ID NO: 27) 5B1YTFTSYWMH (SEQ ID IGEINPRDGVSNCN TIWEDYFDY NO: 21) (SEQ ID NO: 24)(SEQ ID NO: 28) 6D8 YTFTSYWMH (SEQ ID IGEIDPSDSYTYYN AAYYSNYVRAY NO: 21)(SEQ ID NO: 25) (SEQ ID NO: 29) 7H12 YTFTSYWMH (SEQ ID IGEINPRDGVSNCNTIWEDYFDY NO: 21) (SEQ ID NO: 24) (SEQ ID NO: 28) 5C6 GYTFTDYNIHYIYPYNGDTG GNYLYYYAMDY (SEQ ID NO: 178) (SEQ ID NO: 180)(SEQ ID NO: 182) 12B12 GFTFSNYGMS TINSNGGRTY YYRYDSYAMDY(SEQ ID NO: 179) (SEQ ID NO: 181) (SEQ ID NO: 183) 17C2 GYTFTDYNIHYIYPYNGDTG GNYLYYYAMDY (SEQ ID NO: 178) (SEQ ID NO: 180)(SEQ ID NO: 182)

TABLE 4AEU or Kabat light chain Framework sequences of anti-Siglec-9 antibodiesAb VL FR1 VL FR2 VH FR3 VL FR4 2D4 DTQMTQSPSSLSA WLQQKPDGTIKRLIFGVPKRFSGSRSGSDYSLT FGGGTKLETK SLGERVSLTC (SEQ ID NO:35)TSSLESEDFADYYC (SEQ (SEQ ID NO: 44) (SEQ ID NO: 30) ID NO: 39) 2D5DVVMTQTPLTLS WLLQRPGQSPKRLIY GVPDRFTGSGSGTDFTL FGGGTKLEIK VTIGQPASTSC(SEQ ID NO: 36) KISRVEAEDLGVYYC (SEQ ID NO: 44) (SEQ ID NO: 31)(SEQ ID NO: 40) 5B1 NVVMTQTPLTLS WLLQRPGQSPKLLIY GVPDRFSGSGSGTDFTLFGAGTKLELK VTLGQPASISC (SEQ ID NO: 37) KINRVEAEDLGVYYC (SEQ ID NO: 45)(SEQ ID NO: 32) (SEQ ID NO: 41) 6D8 DIQKPQSPFYMC GSAEKPEKPFKLEIYGAPSRFSGRGSGTDFSLT FGGGTKLEIK VSGGETISISS (SEQ ID NO: 38)INRGESEDCAEYYC (SEQ (SEQ ID NO: 44) (SEQ ID NO: 33) ID NO: 42) 7H12NVZMTQTPLTLS WLLQRPGQSPKLLIY GVPDRFSGSGSGTDFTL FGGGTKLEMK VTLGQPASISC(SEQ ID NO: 37) KINRVEAEDLGVYFC (SEQ ID NO: 46) (SEQ ID NO: 34)(SEQ ID NO: 43) 5C6 DVVMTQTPLSLP WYLQKSGQSPKLLIY GVPDRFRGSGSGTDFTLFGAGTKLELK VSLGDQVSISC (SEQ ID NO: 186) KISRVEAEDLGVYFC (SEQ ID NO: 45)(SEQ ID NO: 184) (SEQ ID NO: 188) 12B12 DIVLTQSPASLAV WYQQKPGQPPKLLIYGIPARFSGSGSRTDFTLTI FGGGTKLEIK SLGQRATISC (SEQ ID NO: 187)NPVEADDVATYYC (SEQ (SEQ ID NO: 44) (SEQ ID NO: 185) ID NO: 189) 17C2DVVMTQTPLSLP WYLQKSGQSPKLLIY GVPDRFRGSGSGTDFTL FGAGTKLELK VSLGDQVSISC(SEQ ID NO: 186) KISRVEAEDLGVYFC (SEQ ID NO: 45) (SEQ ID NO: 184)(SEQ ID NO: 188)

TABLE 4BEU or Kabat heavy chain Framework sequences of anti-Siglec-9 antibodiesAb VH FR1 VH FR2 VH FR3 VH FR4 2D4 EVKLVESGGALV WVRQTPEKRLEW (SEQDSVKGRFAISRDNAKNT WGQGTTLTVS KPGGSLKLSCAAS ID NO: 51) LYLQMSNLRSEDTALYYS (SEQ ID G (SEQ ID NO: 47) C (SEQ ID NO: 54) NO: 58) 2D5 QVQLQQSGPELVWVKQRPGKGLEW GKFKGKATLTADKSSST WGQGTLVTVS KPGASVKISCKAS (SEQ ID NO: 52)AYMQLSSLTSEDSAVYF A (SEQ ID G (SEQ ID NO: 48) C (SEQ ID NO: 55) NO: 59)5B1 QVQLQQSGAEVV WVKQRPGQGLEW EKFTSKATLTVDTSSNT WGQGTTLTVS KPGASVKLSCKA(SEQ ID NO: 53) AYMQLNNLTSEDSAVY T (SEQ ID FG (SEQ ID NO: 49)YC (SEQ ID NO: 56) NO: 60) 6D8 VQLQQSGAELVK WVKQRPGQGLEWQKFKGKATLTVDKSSST WGQGTLVTVS PGASVKLSCKAS (SEQ ID NO: 53)AYMQLSSLTSEDSAVYY A (SEQ ID G (SEQ ID NO: 50) C (SEQ ID NO: 57) NO: 59)7H12 QVQLQQSGAEVV WVKQRPGQGLEW EKFTSKATLTVDTSSNT WGQGTTLTVS KPGASVKLSCKA (SEQ ID NO: 53) AYMQLNNLTSEDSAVY T (SEQ ID FG (SEQ ID NO: 49)YC (SEQ ID NO: 56) NO: 60) 5C6 EVQLQQSGPELV WVKQSQGKSLEWIGYNQKFQNKATLTVDNSS WGQGTSVTVS KPGASVRISCKAS (SEQ ID NO: 192)STAYMELRSLTSEDSAV S (SEQ ID (SEQ ID NO: 190) YYCAN (SEQ ID NO: 194)NO: 196) 12B12 EVQLVESGGGLV WVRQILDKRLELVA YPDSVKGRFTISRDNAK WGQGTSVTVSQPGGSLKLSCAAS (SEQ ID NO: 193) NTLYLQMSSLRSEDTAIY S (SEQ ID(SEQ ID NO: 191) YCVT (SEQ ID NO: 195) NO: 196) 17C2 EVQLQQSGPELVWVKQSQGKSLEWIG YNQKFQNKATLTVDNSS WGQGTSVTVS KPGASVRISCKAS(SEQ ID NO: 192) STAYMELRSLTSEDSAV S (SEQ ID (SEQ ID NO: 190)YYCAN (SEQ ID NO: 194) NO: 196)

Characterization of Siglec-9 Antibody Binding

Initial characterization of Siglec-9 antibodies involved determiningtheir ability to bind Siglec-9 expressed on human primary monocytes,human primary macrophages, human primary dendritic cells, human primaryneutrophils, and human primary natural killer (NK) cells. Cells wereharvested, plated at 10⁶/ml in a 96 well plate, and incubated in 100 ulPBS containing 2% FBS, 2 mM EDTA and 10 μg/ml Mab and Fc blockingreagent for 1 hour in ice. Cells were washed twice and incubated in 100ul PBS containing 2% FBS, 2 mM EDTA and 5 μg/ml PE-conjugated secondaryantibody for 30 minutes on ice. Cells were washed twice in cold PBS andanalyze by flow cytometry on a BD FACS Canto. Data analysis andcalculation of MFI values was performed with FlowJo (TreeStar) softwareversion 10.0.7.

FACS staining analysis indicates that Siglec-9 is expressed on primarymyeloid and lymphoid cells, including human primary monocytes,macrophages, dendritic cells, neutrophils, and NK cells (FIG. 4).

Table 5 demonstrates antibody binding to human primary cells expressingSiglec-9. Percent positive binding and mean fluorescent intensity (MFI)values for cell types bound by Siglec-9 antibodies are listed in Table5. Binding is compared to an isotype control. The antibodies hind tohuman primary monocytes, human primary dendritic cells, and humanprimary macrophages. In Table 5, “Buffer” refers to cells that weretreated only with the PBS buffer described supra; “Secondary Ab” refersto cells that were treated only with the PE-conjugated secondaryantibody; and “mIgG1,” “mIgG2a,” and “mIgG2b” refer to isotype controlantibodies. FIG. 5 demonstrates antibody binding to human primarydendritic cells.

TABLE 5 Siglec-9 antibody binding to human primary cells MonocytesDendritic Cells Macrophages Antibody % Positive MFI % Positive MFI %Positive MFI 2D4 95.0 538 99.3 757 99.10 849 2D5 9.1 145 34.2 252 93.20692 5B1 75.9 310 97.8 646 98.70 621 6B2 93.6 430 69.2 412 99.00 932 6D829.6 198 27.6 197 93.50 473 7H12 38.3 216 60.5 331 94.80 467 ControlsBuffer 0.1 63 1.62 67.4 0.39 74 Secondary Ab 2.6 105 7.05 90.1 3.58 153mIgG1 2.6 104 12.3 132 3.01 115 mIgG2a 3.5 127 8.45 121 22.60 164 mIgG2b4.0 145 26.4 319 12.60 180

The binding affinity of each anti-Siglec-9 antibody was determined bymeasuring their K_(D) by surface plasmon resonance (SPR) assays. SPRdata was collected at a rate of 10 Hz at 25° C. on a BiaCore T200instrument. Data analysis was performed using BiaCore T200 EvaluationSoftware, version 2.0. HBS-EP+ (100 mM HEPES, 1.5 M NaCl, 30 mM EDTA,0.5% v/v Surfactant P20, pH 7.4) was used as running buffer and forpreparing reagents.

Mouse-derived antibodies (25 nM) against Siglec-9 were captured (60 scontact time, 30 uL/min flow rate, 0 s stabilization time) on a CM5sensor chip (GE Healthcare) immobilized with anti-mouse IgG.Histidine-tagged human Siglec-9 (NovoProtein) was then flowed over thecaptured anti-Siglec-9 surface (120 s contact time, 30 uL/min flow rate,300 s dissociation time). Concentration of antigen ranged from 5 nM to100 nM, depending on information obtained from an initial screen.Duplicate single-concentration trials were performed and bracketed by ablank (0 nM antigen) sample. The chip surface was regenerated in betweencycles using 10 mM glycine-HCl, pH 1.7 (60 s contact time, 30 uL/minflow rate, 60 s stabilization time). The resulting SPR signal wasobtained as the difference in response from measurements performed on ablank flow cell.

Single-concentration kinetic analysis (Canziani, et al. (2004)Analytical Biochemistry 325:301-307) was performed using a 1:1interaction model to extract association and dissociation rate constants(k_(a) and k_(d), respectively) for each antibody. Affinity constants(K_(D)) were calculated from the ratio k_(d)/k_(a). We validated thesingle-concentration analysis with a multiple-concentration approach(five concentrations of antigen used, plus one blank).

The results are listed in Table 6A. FIG. 6A shows SPR sensogramsdepicting antibody affinity.

TABLE 6A Siglec-9 antibody affinities Antibody mAb nM Siglec-9 nM K_(D)2D4 25 25 0.3 nM 2D5 25 100 5.1 nM 5B1 25 100 2.7 nM 6B2 25 200 6.5 nM6D8 25 200 6.7 nM 7H12 25 200 9.0 nM

The binding affinity of anti-Siglec-9 antibodies 2D4, 5C6, and 12B12 wasalso determined by measuring their K_(D) using a Fortebio Octet RED96system, with all steps performed in 0.25% casein in PBS. Antibodies werecaptured on anti-mouse IgG Fc capture (AMC) biosensor tips, after whicha baseline was run for 60 seconds. A 15 minute association step wasperformed in a dilution series of His-tagged Siglec 9 antigen, rangingfrom 3.13 nM to 50 nM, after which dissociation was measured over 60minutes. Affinity constants (K_(D)) were calculated from the ratiok_(d)/k_(a).

The Fortebio Octet RED96results are listed in Table 6B. FIG. 6B showssensograms depicting antibody affinity.

TABLE 6B Siglec-9 antibody affinity Antibody Assay K_(a) (1/Ms) _(Kd)(1/S) K_(D) (nM) 2D4 1 7.55E⁺⁰⁴ 1.22E⁻⁰⁵ 1.62E⁻¹⁰ 2D4 2 7.52E⁺⁰⁴2.21E⁻⁰⁵ 2.94E⁻¹⁰ 2D4 Avg. 7.54E⁺⁰⁴ 1.72E⁻⁰⁵ 0.23 nM 5C6 1 2.31E⁺⁰⁵1.23E⁻⁰⁴ 5.32E⁻¹⁰ 5C6 2 2.09E⁺⁰⁵ 1.16E⁻⁰⁴ 5.57E⁻¹⁰ 5C6 Avg. 2.20E⁺⁰⁵1.20E⁻⁰⁴ 0.54 nM 12B12 1 1.17E⁺⁰⁵ 7.45E⁻⁰⁵ 6.39E⁻¹⁰ 12B12 2 1.12E⁺⁰⁵9.85E⁻⁰⁵ 8.79E⁻¹⁰ 12B12 Avg. 1.14E⁺⁰⁵ 8.65E⁻⁰⁵ 0.76 nM

Antibody Humanization

Antibody humanization is used to transform antibodies generated in adifferent species to best resemble a human antibody through sequence andstructural relationships in order to prevent immunogenicity in humanadministration. Antibodies from different species share characteristicsequence and structural features that allow the grafting of thespecificity-determining regions (SDRs) of the non-human antibody onto ahuman antibody framework. This results in retention of the specificityof the non-human antibody. The humanization process involvesidentification of the non-human antibody sequence and features,including the framework regions and SDRs. The following criteria areused to humanize an antibody: 1) percent similarity in framework regionsbetween non-human and known human antibodies, 2) length similarity inSDRs between non-human and known human antibodies, 3) genes used togenerate the framework regions of the human antibody, and 4) previoususe of human antibody frameworks in humanizations and as therapeutics.Similarity in framework regions and SDR lengths are important becausedifferences can generate structural differences in the antibody that canalter the specificity of the antibody. Specific genes used to generatethe framework of human antibodies are known to be beneficial ordetrimental to the stability or specificity of the antibody and areselectively used or avoided, accordingly. Lastly, previously successfulhumanization frameworks, including those used in human therapeutics,which are well tolerated with good half-lives, are likely candidates forfuture successful humanizations.

As shown in Tables 7A and 7B, 10 humanized light chain and 10 humanizedheavy variable region sequences were identified for each of theantibodies 2D4, 2D5, 5B 1, 6D8, and 7H12; three light chain variableregions sequences and four heavy chain variable region sequences wereidentified for antibody 5C6; and three light chain variable regionssequences and two heavy chain variable region sequences were identifiedfor antibody 12B 12. In Tables 7A and 7B, bolded letters indicate HVRsequences.

TABLE 7A Humanized light chain variable regions Antibody variantHumanized sequences Antibody 2D4 2D4 Wild-typeDIQMTQSPSSLSASLGERVSLTCRASQEISGYLGWLQQKPDGTIKRLIFSTSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPPTFGGGTKLEIK (SEQ ID NO: 61)2D4V1-9 DIQLTQSPSFLSASVGDRVTITCRASQEISGYLGWYQQKPGKAPKWYSTSTLDSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 62)2D4V1-39 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLGWYQQKPGKAPKLLIYSTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 63)2D4V1-5 DIQMTQSPSTLSASVGDRVTITCRASQEISGYLGWYQQKPGKAPKLLIYSTSTLDSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 64)2D4V1-33 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLGWYQQKPGKAPKLLIYSTSTLDSGVPSRFSGSGSGTDFTFTISSLQPEDTATYYCLQYASYPPTFGQGTKVETK (SEQ ID NO: 65)2D4V3-15 EIVMTQSPATLSVSPGERATLSCRASQEISGYLGWYQQKPGQAPRLLIYSTSTLDSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 66)2D4V3-11 EIVLTQSPATLSLSPGERATLSCRASQEISGYLGWYQQKPGQAPRLLIYSTSTLDSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 67)2D4V3-20 EIVLTQSPGTLSLSPGERATLSCRASQEISGYLGWYQQKPGQAPRLLIYSTSTLDSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 68)2D4V2-30 DVVMTQSPLSLPVTLGQPASISCRASQEISGYLGWFQQRPGQSPRRLIYSTSTLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 69)2D4V4-1 DIVMTQSPDSLAVSLGERATINCRASQEISGYLGWYQQKPGQPPKLLIYSTSTLDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 70)2D4V2-28 DIVMTQSPLSLPVTPGEPASISCRASQEISGYLGWYLQKPGQSPQLLIYSTSTLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQYASYPPTFGQGTKVEIK (SEQ ID NO: 71)Antibody 2D5 2D5 Wild-typeDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVCKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK (SEQ ID NO: 72)2D5V2-30 DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVCKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTKVEIK (SEQ ID NO: 73) 2D5V2-28DIVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVCKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTKVEIK (SEQ ID NO: 74)2D5V4-1 DIVMTQSPDSLAVSLGERATINCKSSQSLLDSDGKTYLNWYQQKPGQPPKWYLVCKLDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCWQGTHFPQTFGQGTKVEIK (SEQ ID NO: 75)2D5V1-5 DIQMTQSPSTLSASVGDRVTITCKSSQSLLDSDGKTYLNWYQQKPGKAPKWYLVCKLDSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCWQGTHFPQTFGQGTKVEIK (SEQ ID NO: 76)2D5V1-39 DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSDGKTYLNWYQQKPGKAPKWYLVCKLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCWQGTHFPQTFGQGTKVEIK (SEQ ID NO: 77)2D5V1-9 DIQLTQSPSFLSASVGDRVTITCKSSQSLLDSDGKTYLNWYQQKPGKAPKWYLVCKLDSGVPSRFSGSGSGTEPTLTISSLQPEDFATYYCWQGTHFPQTEGQGTKVEIK (SEQ ID NO: 78)2D5V1-33 DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSDGKTYLNWYQQKPGKAPKWYLVCKLDSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCWQGTHFPQTFGQGTKVEIK (SEQ ID NO: 79)2D5V3-11 EIVLTQSPATLSLSPGERATLSCKSSQSLLDSDGKTYLNWYQQKPGQAPRLLIYLVCKLDSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCWQGTHFPQTFGQGTKVEIK (SEQ ID NO: 80)2D5V3-15 EIVMTQSPATLSVSPGERATLSCKSSQSLLDSDGKTYLNWYQQKPGQAPRLLIYLVCKLDSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCWQGTHFPQTFGQGTKVEIK (SEQ ID NO: 81)2D5V3-20 EIVLTQSPGTLSLSPGERATLSCKSSQSLLDSDGKTYLNWYQQKPGQAPRLLIYLVCKLDSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCWQGTHFPQTFGQGTKVETK (SEQ ID NO: 82)Antibody 5B1 5B1 Wild-typeNVVMTQTPLTLSVTLGQPASISCKSSQSLLYTNGKTYLNWLLQRPGQSPKWYLVSKLESGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYCLQATHFPLTFGAGTKLELK (SEQ ID NO: 83)5B1V2-30 DVVMTQSPLSLPVTLGQPASISCKSSQSLLYTNGKTYLNWFQQRPGQSPRRLIYLVSKLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQATHFPLTFGQGTKVEIK (SEQ ID NO: 84)5B1V2-28 DIVMTQSPLSLPVTPGEPASISCKSSQSLLYTNGKTYLNWYLQKPGQSPQLLIYLVSKLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQATHFPLTFGQGTKVEIK (SEQ ID NO: 85)5B1V4-1 DIVMTQSPDSLAVSLGERATINCKSSQSLLYTNGKTYLNWYQQKPGQPPKWYLVSKLESGVPDRESGSGSGTDETLTISSLQAEDVAVYYCLQATHFPLTEGQGTKVEIK (SEQ ID NO: 86)5B1V1-5 DIQMTQSPSTLSASVGDRVTITCKSSQSLLYTNGKTYLNWYQQKPGKAPKWYLVSKLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCLQATHFPLTFGQGTKVEIK (SEQ ID NO: 87)5B1V1-39 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTNGKTYLNWYQQKPGKAPKWYLVSKLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQATHFPLTEGQGTKVEIK (SEQ ID NO: 88)5B1V1-9 DIQLTQSPSFLSASVGDRVTITCKSSQSLLYTNGKTYLNWYQQKPGKAPKWYLVSKLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQATHFPLTFGQGTKVEIK (SEQ ID NO: 89)5B1V1-33 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTNGKTYLNWYQQKPGKAPKWYLVSKLESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQATHFPLTFGQGTKVETK (SEQ ID NO: 90)5B1V3-11 EIVLTQSPATLSLSPGERATLSCKSSQSLLYTNGKTYLNWYQQKPGQAPRLLIYLVSKLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQATHFPLTFGQGTKVEIK (SEQ ID NO: 91)5B1V3-15 EIVMTQSPATLSVSPGERATLSCKSSQSLLYTNGKTYLNWYQQKPGQAPRLLIYLVSKLESGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQATHFPLTFGQGTKVEIK (SEQ ID NO: 92)5B1V3-20 EIVLTQSPGTLSLSPGERATLSCKSSQSLLYTNGKTYLNWYQQKPGQAPRLLIYLVSKLESGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCLQATHFPLTFGQGTKVEIK (SEQ ID NO: 93)Antibody 6D8 6D8 Wild-typeDIQKPQSPFYMCVSGGETISISSHTTRGIYWYKGGSAEKPEKPFKLEIYCARNGEGGAPSRFSGRGSGTDFSLTINRGESEDCAEYYCAQFYQFPWTFGGGTKLEIK (SEQ ID NO: 94)6D8V1-33 DIQMTQSPSSLSASVGDRVTITCHTTRGIYWYKGWYQQKPGKAPKLLIYCARNGEGGVPSRFSGSGSGTDUTFTISSLQPEDIATYYCAQFYQFPWTEGQGTKVEIK (SEQ ID NO: 95)6D8V1-9 DIQLTQSPSFLSASVGDRVTITCHTTRGIYWYKGWYQQKPGKAPKWYCARNGEGGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCAQFYQFPWTFGQGTKVEIK (SEQ ID NO: 96)6D8V1-39 DIQMTQSPSSLSASVGDRVTITCHTTRGIYWYKGWYQQKPGKAPKLLIYCARNGEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQFYQFPWTFGQGTKVEIK (SEQ ID NO: 97)6D8V3-11 EIVLTQSPATLSLSPGERATLSCHTTRGIYWYKGWYQQKPGQAPRLLIYCARNGEGGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCAQFYQFPWTFGQGTKVEIK (SEQ ID NO: 98)6D8V1-5 DTQMTQSPSTLSASVGDRVTITCHTTRGIYWYKGWYQQKPGKAPKLLIYCARNGEGGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAQFYQFPWTFGQGTKVETK (SEQ ID NO: 99)6D8V3-15 EIVMTQSPATLSVSPGERATLSCHTTRGIYWYKGWYQQKPGQAPRLLIYCARNGEGGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCAQFYQFPWTFGQGTKVEIK (SEQ ID NO: 100)6D8V3-20 EIVLTQSPGTLSLSPGERATLSCHTTRGIYWYKGWYQQKPGQAPRLLIYCARNGEGGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCAQFYQFPWTFGQGTKVEIK (SEQ ID NO: 101)6D8V2-28 DIVMTQSPLSLPVTPGEPASISCHTTRGIYWYKGWYLQKPGQSPQLLIYCARNGEGGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQFYQFPWTFGQGTKVEIK (SEQ ID NO: 102)6D8V4-1 DIVMTQSPDSLAVSLGERATINCHTTRGIYWYKGWYQQKPGQPPKWYCARNGEGGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCAQFYQFPWTFGQGTKVEIK (SEQ ID NO: 103)6D8V2-30 DVVMTQSPLSLPVTLGQPASISCHTTRGIYWYKGWFQQRPGQSPRRLIYCARNGEGGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQFYQFPWTFGQGTKVEIK (SEQ ID NO: 104)Antibody 7H12 Antibody 7H12 Wild-typeNVZMTQTPLTLSVTLGQPASISCKSSQSLLYTNGKTYLNWLLQRPGQSPKWYLVSKLESGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCLQAPHFPLTFGGGTKLEMK (SEQ ID NO: 105)7H12V2-30 DVVMTQSPLSLPVTLGQPASISCKSSQSLLYTNGKTYLNWFQQRPGQSPRRLIYLVSKLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQAPIIFPLTFGQGTKVEIK(SEQ ID NO: 106) 7H12V2-28DIVMTQSPLSLPVTPGEPASISCKSSQSLLYTNGKTYLNWYLQKPGQSPQLLIYLVSKLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQAPHFPLTFGQGTKVEIK (SEQ ID NO: 107)7H12V4-1 DTVMTQSPDSLAVSLGERATINCKSSQSLLYTNGKTYLNWYQQKPGQPPKLLTYLVSKLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCLQAPHFPLTFGQGTKVEIK(SEQ ID NO: 108) 7H12V1-5DIQMTQSPSTLSASVGDRVTITCKSSQSLLYTNGKTYLNWYQQKPGKAPKWYLVSKLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCLQAPHFPLTFGQGTKVEIK(SEQ ID NO: 109) 7H12V1-9DIQLTQSPSFLSASVGDRVTITCKSSQSLLYTNGKTYLNWYQQKPGKAPKWYLVSKLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQAPHFPLTEGQGTKVEIK (SEQ ID NO: 110)7H12V1-33 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTNGKTYLNWYQQKPGKAPKLLIYLVSKLESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQAPHFPLTFGQGTKVEIK(SEQ ID NO: 111) 7H12V1-39DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTNGKTYLNWYQQKPGKAPKLLIYLVSKLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQAPHFPLTFGQGTKVEIK(SEQ ID NO: 112) 7H12V3-11EIVLTQSPATLSLSPGERATLSCKSSQSLLYTNGKTYLNWYQQKPGQAPRLLIYLVSKLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQAPHFPLTEGQGTKVEIK (SEQ ID NO: 113)7H12V3-15 EIVMTQSPATLSVSPGERATLSCKSSQSLLYTNGKTYLNWYQQKPGQAPRLLIYLVSKLESGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQAPHFPLTFGQGTKVEIK(SEQ ID NO: 114) 7H12V3-20EIVLTQSPGTLSLSPGERATLSCKSSQSLLYTNGKTYLNWYQQKPGQAPRLLIYLVSKLESGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCLQAPHFPLTEGQGTKVEIK (SEQ ID NO: 115)Antibody 5C6 Antibody 5C6 Wild-typeDVVMTQTPLSLPVSLGDQVSISCSSSQSLVHSNGNTYLHWYLQKSGQSPKLLIYKVSNRFSGVPDRFRGSGSGTDFTLKISRVEAEDLGVYFCSQNTIIVPLTFGAGTKLELK(SEQ ID NO: 197) 5C6-L1DVVMTQSPLSLPVTLGQPASTSCSSSQSLVHSNGNTYLHWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPLTFGQGTKLEIK (SEQ ID NO: 198)5C6-L2 DVVMTQSPLSLPVTLGQPVSISCSSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPLTFGQGTKLEIK (SEQ ID NO: 199)5C6-L3 DVVMTQSPLSLPVTLGQPVSISCSSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCSQNTHVPLTEGQGTKLELK (SEQ ID NO: 200)Antibody 12B12 Antibody 12B12 Wild-typeDIVLTQSPASLAVSLGQRATISCRASENVDSYGISFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 201)12B12-L1 DIQMTQSPSSLSASVGDRVTITCRASENVDSYGISFMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPWTFGQGTKLEIK (SEQ ID NO: 202)12B12-L2 DIQLTQSPSSLSASVGDRVTITCRASENVDSYGISFMHWYQQKPGKAPKLLIYRASNLESGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPWTFGQGTKLEIK (SEQ ID NO: 203)12B12-L3 DIQLTQSPSSLSASVGDRATITCRASENVDSYGISFMHWYQQKPGKAPKLLIYRASNLESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPWTFGQGTKLEIK (SEQ ID NO: 204)

TABLE 7B Humanized heavy chain variable regions Antibody variantHumanized sequences Antibody 2D4 2D4 Wild-typeEVKLVESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQTPEKRLEWVATINNGGSYTYYSDSVKGRPAISRDNAKNFLYLQMSNLRSLDFALYYCVRRDYGTSDFDYWGQGTTLTVSS (SEQ ID NO: 116) 2D4V3-23EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATINNGGSYTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 117) 2D4V3-7EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATINNGGSYTYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 118) 2D4V3-30QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATINNGGSYTYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 119) 2D4V3-48EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATINNGGSYTYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 120) 2D4V1-46QVQLVQSGAEVKKPGASVKVSCKASGFTFSNYAMSWVRQAPGQGLEWVATINNGGSYTYYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 121) 2D4V1-69QVQLVQSGAEVKKPGSSVKVSCKASGFTFSNYAMSWVRQAPGQGLEWVATINNGGSYTYYSQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 122) 2D4V5-51EVQLVQSGAEVKKPGESLKISCKGSGFTFSNYAMSWVRQMPGKGLEWVATINNGGSYTYYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 123) 2D4V4-59QVQLQESGPGLVKPSETLSLTCTVSGFTFSNYAMSWIRQPPGKGLEWVATINNGGSYTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 124) 2D4V3-15EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATINNGGSYTYYSAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 125) 2D4V4-39QLQLQESGPGLVKPSETLSLTCTVSGFTFSNYAMSWIRQPPGKGLEWVATINNGGSYTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVRRDYGTSDFDYWGQGTLVTVSS (SEQ ID NO: 126) Antibody 2D5 2D5 Wild-typeQVQLQQSGPELVKPGASVKISCKASGYAFSSYWMNWVKQRPGKGLEWIGRIYPRDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARWLLRFAYWGQGTLVTVSA (SEQ ID NO: 127) 2D5V5-51EVQLVQSGAEVKKPGESLKISCKGSGYAFSSYWMNWVRQMPGKGLEWIGRIYPRDGDTNYNPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 128) 2D5V1-46QVQLVQSGAEVKKPGASVKVSCKASGYAFSSYWMNWVRQAPGQGLEWIGRIYPRDGDTNYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 129) 2D5V1-69QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWIGRIYPRDGDTNYNQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 130) 2D5V3-23EVQLLESGGGLVQPGGSLRLSCAASGYAFSSYWMNWVRQAPGKGLEWIGRIYPRDGDTNYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 131) 2D5V3-48EVQLVESGGGLVQPGGSLRLSCAASGYAFSSYWMNWVRQAPGKGLEWIGRIYPRDGDTNYNDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 132) 2D5V3-7EVQLVESGGGLVQPGGSLRLSCAASGYAFSSYWMNWVRQAPGKGLEWIGRIYPRDGDTNYNDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 133) 2D5V3-30QVQLVESGGGVVQPGRSLRLSCAASGYAFSSYWMNWVRQAPGKGLEWIGRIYPRDGDTNYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 134) 2D5V4-59QVQLQESGPGLVKPSETLSLTCTVSGYAFSSYWMNWIRQPPGKGLEWIGRIYPRDGDTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 135) 2D5V3-15EVQLVESGGGLVKPGGSLRLSCAASGYAFSSYWMNWVRQAPGKGLEWIGRIYPRDGDTNYNAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 136) 2D5V4-30-4QVQLQESGPGLVKPSQTLSLTCTVSGYAFSSYWMNWIRQPPGKGLEWIGRIYPRDGDTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARWLLRFAYWGQGTLVTVSS (SEQ ID NO: 137) Antibody 5B1 5B1 Wild-typeQVQLQQSGAEVVKPGASVKLSCKAFGYTFTSYWMHWVKQRPGQGLEWIGEINPRDGVSNCNEKFTSKATLTVDTSSNTAYMQLNNLTSEDSAVYYCTIWEDYFDYWGQGTTLTVST (SEQ ID NO: 138) 5B1V1-46QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPRDGVSNCNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 139) 5B1V5-51EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYWMHWVRQMPGKGLEWIGEINPRDGVSNCNPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 140) 5B1V1-69QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPRDGVSNCNQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 141) 5B1V3-7EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 142) 5B1V3-30QVQLVESGGGVVQPGRSLRLSCAASGYTFTSYWMIIWVRQAPGKGLEWIGEINPRDGVSNCNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 143) 5B1V3-23EVQLLESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNDSVKGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 144) 5B1V3-48EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 145) 5B1V4-59QVQLQESGPGLVKPSETLSLTCTVSGYTFTSYWMHWIRQPPGKGLEWIGEINPRDGVSNCNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 146) 5B1V3-15EVQLVESGGGLVKPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 147) 5B1V4-39QLQLQESGPGLVKPSETLSLTCTVSGYTFTSYWMHWIRQPPGKGLEWIGEINPRDGVSNCNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 148) Antibody 6D8 6D8 Wild-typeVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEIDPSDSYTYYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAAYYSNYVRAYWGQGTLVTVSA (SEQ ID NO: 149) 6D8V1-46QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEIDPSDSYTYYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 150) 6D8V5-51EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYWMIIWVRQMPGKGLEWIGEIDPSDSYTYYNPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 151) 6D8V3-7EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEIDPSDSYTYYNDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 152) 6D8V3-23EVQLLESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEIDPSDSYTYYNDSVKGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 153) 6D8V3-30QVQLVESGGGVVQPGRSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEIDPSDSYTYYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 154) 6D8V3-48EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEIDPSDSYTYYNDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 155) 6D8V1-69QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEIDPSDSYTYYNQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 156) 6D8V4-59QVQLQESGPGLVKPSETLSLTCTVSGYTFTSYWMHWIRQPPGKGLEWIGEIDPSDSYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 157) 6D8V3-15EVQLVESGGGLVKPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEIDPSDSYTYYNAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 158) 6D8V4-30-4QVQLQESGPGLVKPSQTLSLTCTVSGYTFTSYWMHWIRQPPGKGLEWIGEIDPSDSYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVIYCAAYYSNYVRAYWGQGTLVTVSS (SEQ ID NO: 159) Antibody 7H12 Antibody 7H12 Wild-typeQVQLQQSGAEVVKPGASVKLSCKAFGYTFTSYWMHWVKQRPGQGLEWIGEINPRDGVSNCNEKFTSKATLTVDTSSNTAYMQLNNLTSEDSAVYYCTIWEDYFDYWGQGTTLTVST (SEQ ID NO: 160) 7H12V1-46QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMIIWVRQAPGQGLEWIGEINPRDGVSNCNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 161) 7H12V5-51EVQLVQSGAEVKKPGESLKTSCKGSGYTFTSYWMHWVRQMPGKGLEWIGEINPRDGVSNCNPSFQGQVTTSADKSTSTAYLQWSSLKASDTAMYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 162) 7H12V1-69QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGEINPRDGVSNCNQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 163) 7H12V3-7EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 164) 7H12V3-30QVQLVESGGGVVQPGRSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 165) 7H12V3-23EVQLLESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 166) 7H12V3-48EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNDSVKGRFTISRDNAKNSLILQMNSLRAEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 167) 7H12V4-59QVQLQESGPGLVKPSETLSLTCTVSGYTFTSYWMHWIRQPPGKGLEWIGEINPRDGVSNCNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 168) 7H12V3-15EVQLVESGGGLVKPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEINPRDGVSNCNAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 169) 7H12V4-39QLQLQESGPGLVKPSETLSLTCTVSGYTFTSYWMHWIRQPPGKGLEWIGEINPRDGVSNCNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTIWEDYFDYWGQGTLVTVSS (SEQ ID NO: 170) Antibody 5C6 Antibody 5C6 Wild-typeEVQLQQSGPELVKPGASVRISCKASGYTFTDYNIHWVKQSQGKSLEWIGYIYPYNGDTGYNQKFQNKATLTVDNSSSTAYMELRSLTSEDSAVYYCANGNYLYYYAMDYWGQGTSVTVSS (SEQ ID NO: 205) 5C6-H1QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNIHWVRQAPGQGLEWMGYIYPYNGDTGYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGNYLYYYAMDYWGQGTLVTVSS (SEQ ID NO: 206) 5C6-H2QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNIHWVRQAPGQGLEWMGYIYPYNGDTGYAQKLQGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCANGNYLYYYAMDYWGQGTLVTVSS (SEQ ID NO: 207) 5C6-H3QVQLVQSGAEVKKPGASVKISCKASGYTFTDYNIHWVRQAPGQGLEWIGYIYPYNGDTGYAQKFQGRATMTVDTSTSTAYMELRSLRSDDTAVYYCANGNYLYYYAMDYWGQGTLVTVSS (SEQ ID NO: 208) 5C6-H4QVQLVQSGAEVKKPGASVKISCKASGYTFTDYNIHWVRQAPGQGLEWIGYIYPYNGDTGYAQKFQGRATMTVDNSTSTAYMELRSLRSDDTAVYYCANGNYLYYYAMDYWGQGTLVTVSS (SEQ ID NO: 209) Antibody 12B12 Antibody 12B12 Wild-typeEVQLVESGGGLVQPGGSLKLSCAASGFTFSNYGMSWVRQILDKRLELVATINSNGGRTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAIYYCVTYYRYDSYAMDYWGQGTSVTVSS (SEQ ID NO: 210) 12B12-H1EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVSTINSNGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYYRYDSYAMDYWGQGTLVTVSS (SEQ ID NO: 211) 12B12-H2EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLELVATINSNGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTYYRYDSYAMDYWGQGTLVTVSS (SEQ ID NO: 212)

Humanization of Antibody 5C6

The heavy chain variable region (VH) and light chain variable region(VL) sequences of murine anti-Siglec-9 antibody 5C6 (S9-5C6.3) wascompared to a library of known human germline sequences. The databasesused were IMGT human VH genes (F+ORF, 273 germline sequences) and IMGThuman VLkappa genes (F+ORF, 74 germline sequences).

For the 5C6 antibody VL, human germline IGKV2-30 (allele 1) was chosenas the acceptor sequence and human light chain IGKJ2 (allele 1) joiningregion (IGKJ gene) was chosen from human joining region sequencescompiled at IMGT® the international ImMunoGeneTics Information System®(FIG. 6C). For the 5C6 antibody VH, human germline IGHV1-18 (allele 1)was chosen as the acceptor sequence and the human heavy chain IGHJ4(allele 1) joining region (IGHJ gene) was chosen from human joiningregion sequences compiled at IMGT® the international ImMunoGeneTicsInformation System® (FIG. 6D). Complementarity determining regions(CDRs) for the antibody VL and VH were defined according to the AbMdefinition (AbM antibody modeling software).

Alteration of human germline framework (i.e., non-CDR residues in the VHand VL) positions to corresponding parental murine sequences may berequired to optimize binding of the humanized antibody. Potentialchanges for each humanized sequence are noted in FIGS. 6C and 6D. FIGS.6C and 6D show sequences of humanized versions of anti-Siglec-9 antibody5C6 (5C6.3).

In the VL domain of antibody 5C6, in CDR-L1, Asp30c-Gly30d-Asn30d-Thr30ehas a high potential for deamidation at both Asn followed byisoaspartate formation at position 30c-30d. Post-translationalmodification at this site could affect binding of the antibody totarget. The parental mouse antibody may be analyzed to determine thelevel of post-translational modification (FIG. 6C). Alternativelyantibody 5C6 may be humanized and then in a final step the NG may bemodified, to e.g., SG, QG, or AG, to determine if binding is maintained;separately, the NT may be altered to, e.g., QT or ST. In the CDR-L2,Asn53 has a low potential for deamidation based on sequence andconformation and may show a low level of this post-translationalmodification (FIG. 6C). In CDR-L3, Asn91 has a medium potential fordeamidation based on sequence and conformation and may show a low levelof this post-translational modification (FIG. 6C). Variant VL sequencesbased on the above are listed in Table 7A.

In the VH domain of antibody 5C6 (5C6.3), Asn33 and Asn 96 in the CDR-H1have low potential for deamidation based on sequence and conformationand may show a low level of post-translational modifications (FIG. 6D).In the CDR-H2, Asn54-Gly55 has a low/medium potential for deamidationfollowed by isoaspartate formation (FIG. 6D). Post-translationalmodification at this site may affect binding of the antibody to target.The parental mouse antibody may be analyzed to determine the level ofpost-translational modification. Alternatively 5C6 may be humanized andthen in a final NG may be altered to e.g., QG, to determine if bindingis maintained. Variant VH sequences based on the above are listed inTable 7B.

Humanization of Antibody 12B12

The heavy chain variable region (VH) and light chain variable region(VL) sequences of murine anti-Siglec-9 antibody 12B12 (S9-12B12.2) wascompared to a library of known human germline sequences. The databasesused were IMGT human VH genes (F+ORF, 273 germline sequences) and IMGThuman VLkappa genes (F+ORF, 74 germline sequences).

For the 12B12 antibody VL, human germline IGKV1-39 (allele 1) was chosenas the acceptor sequence and human light chain IGKJ2 (allele 1) joiningregion (IGKJ gene) was chosen from human joining region sequencescompiled at IMGT® the international ImMunoGeneTics Information System®(FIG. 6E). For the 12B12 antibody VH, human germline IGHV3-23 (allele 4)was chosen as the acceptor sequence and the human heavy chain IGHJ4(allele 1) joining region (IGHJ gene) was chosen from human joiningregion sequences compiled at IMGT® the international ImMunoGeneTicsInformation System® (FIG. 6F). Complementarity determining regions(CDRs) for the antibody VL and VH were defined according to the AbMdefinition (AbM antibody modeling software).

Alteration of human germline framework (i.e., non-CDR residues in the VHand VL) positions to corresponding parental murine sequences may berequired to optimize binding of the humanized antibody. Potentialchanges for each humanized sequence are noted in FIGS. 6E and 6F. FIGS.6E and 6F show sequences of humanized versions of anti-Siglec-9 antibody12B12 (12B 12.2).

In the VL domain of antibody 12B12, Asn29, Asn53, and Asn92 in theCDR-L1 have a low potential for deamidation based on sequence andconformation and may show a low level of post-translational modification(FIG. 6E). In the CDR-L3, Trp96 is likely to be at least partiallysolvent-exposed and hence may undergo oxidation under stress conditions(FIG. 6E). Variant VL sequences based on the above are listed in Table7A.

In the VH domain of antibody 12B12 (12B12.2), Asn31 in the CDR-H1 has alow potential for deamidation based on sequence and conformation and mayshow a low level of posttranslational modification, while Asn52 in theCDR-H1 has a medium potential for deamidation based on sequence andconformation and may show a low level of post-translational modification(FIG. 6F). In the CDR-H2, Asn53-Gly54-Gly55 has a medium/high potentialfor deamidation followed by isoaspartate formation (FIG. 6E).Post-translational modification at this site may affect binding of theantibody to target. The parental mouse antibody may be analyzed todetermine the level of post-translational modification. Alternativelyantibody 12B 12 can be humanized and then in a final step the NGG may bealtered to, e.g., QGG or AGG, to determine if binding is maintained.Variant VH sequences based on the above are listed in Table 7B.

Example 2: Epitope Mapping of Siglec-9 Antibodies

Siglec-9 antibodies were tested for their ability to bind 15 or 25-merpeptides spanning the entire human Siglec-9. The Siglec-9 antibodieswere also compared to a reference Siglec-9 antibody by determining theirSiglec-9 binding region.

Methodology

Linear 15-mer peptides were synthesized based on the sequence of humanSiglec-9 (SEQ ID NO: 1), with a 14 residue overlap. In addition, linear25-mer peptides were synthesized based on sequence of human Siglec-9(SEQ ID NO: 1) with a single residue shift. The binding of Siglec-9antibodies to each of the synthesized peptides was tested in an ELISAbased method. In this assay, the peptide arrays were incubated withprimary antibody solution (overnight at 4° C.). After washing, thepeptide arrays were incubated with a 1/1000 dilution of an antibodyperoxidase conjugate (SBA, cat. nr. 2010-05) for one hour at 25° C.After washing, the peroxidase substrate2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μl/ml of 3%H₂O₂ were added. After one hour, the color development was measured. Thecolor development was quantified with a charge coupled device (CCD)camera and an image processing system.

Epitope binning of the antibodies was performed on a Forte Bio OctetRed384 system (Pall Forte Bio Corporation, Menlo Park, Calif.) using astandard sandwich format binning assay (see Estep et al, (2013) MAbs5(2):270-8). Control anti-target IgG was loaded onto AHQ sensors andunoccupied Fc-binding sites on the sensor were blocked with anon-relevant human IgG1 antibody. The sensors were then exposed to 100nM target antigen followed by a second anti-target antibody. Data wasprocessed using ForteBio's Data Analysis Software 7.0. Additionalbinding by the second antibody after antigen association indicates anunoccupied epitope (non-competitor), while no binding indicates epitopeblocking (competitor).

Alternatively, to reconstruct epitopes of the target molecule, librariesof looped and combinatorial peptides were synthesized. An aminofunctionalized polypropylene support was obtained by grafting with aproprietary hydrophilic polymer formulation, followed by reaction witht-butyloxycarbonyl-hexamethylenediamine (BocHMDA) usingdicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) andsubsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA).Standard Fmoc-peptide synthesis was used to synthesize peptides on theamino-functionalized solid support by custom modified JANUS liquidhandling stations (Perkin Elmer).

Synthesis of structural mimics was done using Pepscan's proprietaryChemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPStechnology allows to structure peptides into single loops anddouble-loops. CLIPS templates are coupled to cysteine residues. Theside-chains of multiple cysteines in the peptides are coupled to one ortwo CLIPS templates. For example, a 0.5 mM solution of the mP2 CLIPS(2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20mM, pH 7.8)/acetonitrile (1:3(v/v)). This solution is added onto thepeptide arrays. The CLIPS template will bind to side-chains of twocysteines as present in the solid-phase bound peptides of thepeptide-arrays (455 wells plate with 3 μl wells). The peptide arrays aregently shaken in the solution for 30 to 60 minutes while completelycovered in solution. Finally, the peptide arrays are washed extensivelywith excess of H₂O and sonicated in disrupt-buffer containing 1%SDS/0.1% β-mercaptoethanol in PBS (pH 7.2) at 70° C. for 30 minutes,followed by sonication in H₂O for another 45 minutes. The T3 CLIPS(2,4,6-tris(bromomethyl)pyridine) carrying peptides were made in asimilar way but now with three cysteines.

Looped Peptides: constrained peptides of length 17. Positions 2-16 are15-mers derived from the target sequence. Native Cys residues areprotected by acetamidomethyl group (ACM). Positions 1 and 17 are Cysthat are linked by mP2 CLIPS moieties. Combinatorial peptides(discontinuous mimics): constrained peptides of length 33. Positions2-16 and 18-32 are 15-mer peptides derived from the target sequence withnative Cys residues protected by ACM. Positions 1, 17 and 33 are Cysthat are linked by T3 CLIPS moieties.

The binding of antibody to each of the synthesized peptides is tested ina PEPSCAN-based ELISA. The peptide arrays are incubated with testantibody solution composed of the experimentally optimized concentrationof the test antibody and blocking solution (for example 4% horse serum,5% ovalbumin (w/v) in PBS/1% Tween80). The peptide arrays are incubatedwith the test antibody solution overnight at 4° C. After extensivewashing with washing buffer (1×PBS, 0.05% Tween80), the peptide arraysare incubated with a 1/1000 dilution of an appropriate antibodyperoxidase conjugate for one hour at 25° C. After washing with thewashing buffer, the peroxidase substrate2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μl/ml of 3%H₂O₂ are added. After one hour, the color development is measured. Thecolor development is quantified with a charge coupled device(CCD)—camera and an image processing system.

Results

The Siglec-9 binding region was determined for 3 anti-Siglec-9antibodies. The binding region is listed in Table 8A.

TABLE 8A Siglec-9 antibody binding region Antibody Siglec-9 bindingregion Amino acid region of SEQ ID: 1 6B2 ¹⁰⁷RDARRSDAGR¹¹⁵ 107-115 7H122D4 ¹⁸⁵VSPLDPSTTR¹⁹⁴ 185-194 2D5 ⁶²FREGANTDQDAPVAT⁷⁶ 62-76 and 86-92 and⁸⁶ETRDRFH⁹² 5B1 ⁸⁶ETRDRFHLLGD⁹⁶ and 86-96 and 105-116 ¹⁰⁵IRDARRSDAGR¹¹⁶

As indicated in Table 8A, antibodies 6B2, 7H12, 2D5, and 5B1 showedrobust binding exclusively for peptides within the extracellular IgVdomain of Siglec-9. As indicated in Table 8A, the peptide recognized byantibodies 6B2 and 7H12 corresponds to amino acid residues 107-115 ofSEQ ID NO: 1 and has the amino acid sequence of: RDARRSDAGR. Thepeptides recognized by antibody 2D5 correspond to amino acid residues62-76 and 86-92 of SEQ ID NO: 1 and have the amino acid sequences of:FREGANTDQDAPVAT and ETRDRFH. The peptides recognized by antibodies 5B 1and 6B2 correspond to amino acid residues 86-96 and 105-116 of SEQ IDNO: 1 and have the amino acid sequences of: ETRDRFHLLGD and IRDARRSDAGR.

As further indicated in Table 8A, antibody 2D4 showed robust bindingexclusively for a peptide within the extracellular Ig-like C2-typedomain of Siglec-9. As indicated in Table 8A, the peptide recognized byantibody 2D4 corresponds to amino acid residues 185-194 of SEQ ID NO: 1and has the amino acid sequences of: VSPLDPSTTR.

Functional Mapping

Shotgun mutagenesis epitope mapping of anti-siglec-9 antibodies wasperformed using an alanine-scanning library for the siglec-9 protein. Asiglec-9 expression construct encoding a C-terminal V5 epitope tag wassubjected to high-throughput alanine scanning mutagenesis (outlined inDavidson and Doranz, 2014 Immunology 143, 13-20) to generate acomprehensive mutation library. Each of the residues representing theSiglec-9 extracellular domain was mutated, most to alanine, whilealanine codons were mutated to serine.

The Siglec-9 mutation library clones, arrayed in a 384-well microplate,were transfected individually into HEK-293T cells and allowed to expressfor 22 hours. Antibodies were digested to generate Fabs, after whichcells were incubated with Fabs diluted in 10% normal goat serum (NGS)(Sigma-Aldrich, St. Louis, Mo.). Prior to library screening, primary Fabconcentrations were determined using an independent immunofluorescencetitration curve against cells expressing wild-type Siglec-9 to ensurethat signals were within the linear range of detection. Fabs weredetected using 7.5 μg/ml AlexaFluor488-conjugated secondary antibody(Jackson ImmunoResearch Laboratories, Westgrove, Pa.) in 10% NGS. Cellswere washed twice with PBS −/− and resuspended in Cellstripper (Cellgro,Manassas, Va.) with 0.1% BSA (Sigma-Aldrich, St. Louis, Mo.). In somecases, higher stringency conditions were used, including increased pH,increased temperature, and increased dissociation time. Mean cellularfluorescence was detected using the Intellicyt high throughput flowcytometer (HTFC, Intellicyt, Albuquerque, N. Mex.). Fab reactivitiesagainst each mutant clone were calculated relative to wild-type Siglec-9protein reactivity by subtracting the signal from mock-transfectedcontrols, and normalizing to the signal from wild-type Siglec-9transfected controls.

Mutated residues within critical clones were identified as critical tothe Fab epitope if they did not support reactivity of the test Fab butdid support reactivity of commercially available reference Mab MAB 1139(R&D Systems, Cat #: 191240), or additional anti-Siglec-9 Fabs. Thiscounter-screen strategy facilitated the exclusion of Siglec-9 mutantsthat were locally misfolded or that had an expression defect.

FIG. 1B depicts the mean binding reactivities and ranges for allcritical residues identified in the screens. Primary critical residueswere identified as residues where mutations were negative for testantibody binding (<30% of binding to WT) but positive for the controlantibody (>80% WT). FIG. 1C-1E depict crystal structure models ofSiglec-9 (PDB ID 2ZG2; Zhuravleva et al., 2008) indicating the criticalresidues as red spheres for anti-Siglec-9 antibodies 2D4, 5C6, and 12B12. The amino acid residues critical for antibody binding are listed inTable 8B.

TABLE 8B Residues involved in anti-Siglec-9 antibody binding AntibodyCritical Siglec-7 residues 2D4 Fab D₁₈₉; P₁₉₀; and R₁₉₄ 12B12 Fab H₄₈;W₅₀; I₅₁; Y₅₂; and I₁₂₆ 5C6 Fab L₂₂; H₄₈; W₅₀; I₅₁; Y₅₂; and K₁₂₃

As indicated in Table 8B, the critical Siglec-9 residues involved inbinding by antibody 2D4 corresponded to amino acid residues D₁₈₉, P₁₉₀,and R₁₉₄ of SEQ ID NO: 1. The critical Siglec-9 residues involved inbinding by antibody 12B12 corresponded to amino acid residues H₄₈, W₅₀,I₅₁, Y₅₂, and I₁₂₆ of SEQ ID NO: 1. The critical Siglec-9 residuesinvolved in binding by antibody 5C6 corresponded to amino acid residuesL₂₂, H₄₈, W₅₀, I₅₁, Y₅₂, and K₁₂₃ of SEQ ID NO: 1.

Example 3: Siglec-9 Antibody-Induced Decrease in Cell Surface Levels ofSiglec-9 In Vitro and In Vivo

In Vitro Expression of Siglec-9

The purpose of the following Example was to test whether anti-Siglec-9and/or Siglec-9 bispecific antibodies reduce the cell surface level ofSiglec-9 on monocytes, macrophages, neutrophils, NK cells, dendriticcells, T cells, and/or microglia.

The ability of anti-Siglec-9 antibodies to reduce cell surface levels ofSiglec-9 on the acute monocytic leukemia cell line THP-1, as well ashuman primary monocytes, human primary dendritic cells (DC) derived fromperipheral blood monocytes, human primary macrophages, and human primarymicroglia was evaluated. Cell samples were plated in 24-well plates at200,000 cells per ml or in 6-well dishes at 500,000 cells in 2 ml ofRPMI supplemented with 10% Hyclone FBS, 2 mM glutamine, pen/strep, andnon-essential amino acids. Siglec-9 antibodies or control isotypes wereadded at 1.0 μg/ml, and incubated for 24 hours at 37° C. with 5% CO₂.

To assess receptor dynamics, antibodies were allowed to bind cells forone hour, washed out and surface levels of Siglec-9 were determined 24and 48 hours later.

Cell surface receptor expression was detected by FACS analysis. Cellswere incubated with anti-Siglec-9-FITC clone HIM3-4, as well as acontrol surface marker (U937: Siglec-5, human monocytes: CD14, humandendritic cells: CD11c, human macrophages: CD11 b) for 30 minutes on icein the dark. Cells were washed 2× in FACS buffer (PBS+2% FBS, 2 mM EDTA)and flow cytometry was performed on a BD FACS Canto. Data was analyzedusing TreeStar FlowJo software. Data was calculated as a percent ofreceptor expression in the absence of antibody using MFI values for therespective fluorophores.

Table 9A and 9B depict the results of Siglec-9 cell surface levels fromhuman cells. In Table 9A and 9B, “No Ab” refers to cells that were thatwere not treated with an antibody; and “mIgG1,” “mIgG2a,” and “mIgG2b”refer to isotype control antibodies.

TABLE 9A Siglec-9 antibodies reduce cell surface level of Siglec-9 in ahuman cell line and in human primary cells Percent Siglec-9 SurfaceExpression THP-1 cells Monocytes DCs % % % % % % Ab Siglec-9 controlSiglec-9 control Siglec-9 control 2D4 56.2 96.0 5.4 60.3 16.6 93.2 2D581.1 95.0 84.4 81.8 94.3 102.2 5B1 61.6 91.1 31.2 62.4 39.2 98.1 6B260.3 95.6 16.9 62.6 27.4 88.2 6D8 62.6 96.7 42.2 68.7 60.2 90.3 7H1268.9 92.8 48.4 72.9 66.2 90.6 No Ab 100 100 100 100 100 100 mIgG1 10099.5 100.5 97.8 96.5 100.3 mIgG2a 95.9 95.9 101.1 95.2 98.6 96.6 mIgG2b98.0 96.0 98.9 103.7 93.4 93.5

TABLE 9B Siglec-9 antibodies reduce cell surface level of Siglec-9 inhuman primary cells Percent Siglec-9 Surface Expression MacrophagesMicroglia Ab % Siglec-9 % control % Siglec-9 % control 2D4 14.0 70.312.8 91.9 2D5 94.6 77.6 90.6 89.3 5B1 24.8 67.7 27.5 102.6 6B2 17.6 64.925.8 105.3 6D8 21.3 68.7 45.4 95.3 7H12 34.2 67.5 54.7 88.8 No Ab 100100 100 100 mIgG1 99.3 95.3 90.3 85.6 mIgG2a 97.8 100.7 91.2 90.1 mIgG2b97.2 93.9 91.7 85.7

As shown in Table 9A and 9B, a majority of the antibodies were able todecrease cell surface levels of Siglec-9 on multiple types of humancells. However, using a threshold value of 80% or higher cell surfaceexpression levels of Siglec-9, it was found that antibody 2D5 does notdecrease cell surface levels of Siglec-9 on any of the tested humancells.

FIG. 7A-7E further demonstrates that a majority of the antibodies wereable to decrease cell surface levels of Siglec-9 on THP-1 cells (FIG.7A), human primary monocytes (FIG. 7B), human primary microglia (FIG.7C), human primary macrophages (FIG. 7D), and human primary dendriticcells (FIG. 7E). FIG. 7F demonstrates that surface levels of Siglec-9 onhuman primary dendritic cells are decreased in a concentration-dependentmanner when treated with Siglec-9 antibodies 2D4 and 6B2.

Additionally, in vitro surface Siglec-9 downregulation studies wereperformed in which Siglec-9 antibodies 5C6, 12B12, and 17C2, and isotypecontrol antibody (mIgG2a) were titrated 2-fold. Primary humanmacrophages were harvested and plated as described above. Siglec-9antibodies were titrated 10-fold with a concentration range of 1 nM to0.001 μg/mL to assess binding. Antibodies were serially diluted andincubated with cells as described above. As shown in FIG. 7I,anti-Siglec-9 antibodies 5C6, 12B 12, and 17C2 were capable ofdownregulating cell surface expression of Siglec-9 on primary humanmacrophages. Cell surface expression of control receptor CD11b was notaffected by treatment with Siglec-9 antibodies (FIG. 7J).

In Vivo Expression of Siglec-9

To test the ability of Siglec-9 antibodies to reduce cell surface levelof Siglec-9 in vivo, humanized NSG mice (hu-NSG) were utilized. Hu-NSGmice are NOD-scid IL2Rγnull mice engrafted with human CD34+hematopoietic stem cells. Female Hu-NSG mice were purchased from Jax andutilized 15 weeks after engraftment with human cells. Mice received anintraperitoneal injection of 40 mg/kg anti-Siglec-9 antibody 2D4 or anisotype control mouse IgG1 antibody (clone MOPC-21) at day 0. At day 1,blood samples were drawn from mice into heparin and processed for FACSanalysis. Briefly, blood samples were first incubated for 5 minutes inice-cold ACK lysis buffer to lyse red blood cells and then washedextensively with cold PBS. This procedure was repeated twice. Cells werethen incubated in FACS buffer (PBS+2% FBS, 2 mM EDTA) in the presence ofanti-human-CD45-Pe-Cy7, anti-mouse-CD45-APC-Cy7,anti-human-CD3-PerCP-Cy5.5, anti-human-CD14-FITC, anti-human-CD11c-PB,anti-Siglec-9-APC, anti-CD33-PE, and a viability die (Life Technologies,Cat # L34957) for 30 min. on ice in the presence of Fc block solution,then washed twice with cold FACS buffer. 4% PFA-fixed samples were thenacquired. Data were acquired on a BD FACS CANTO™ II cytometer (BectonDickinson) and analyzed with FlowJo software. The level of expression ofSiglec 9 and control receptor CD33 was determined in a hCD45+, hCD14+cell population at day −7, 1, 7, 14, and 21 post antibody treatment.

As shown in FIGS. 7G and 7H, treatment with the anti-Siglec-9 antibody2D4 was able to decrease cell surface levels of Siglec-9 in cells ofperipheral blood of the treated hu-NSG mice, when compared to controlmIgG1 antibody treatment. Siglec-9 expression was decreased by ˜80% asearly as 1 day post antibody treatment (FIG. 7G). As a comparison, cellsurface expression of the unrelated surface receptor CD33 did notdecrease significantly after anti-Siglec-9 antibody treatment, ascompared to cell surface levels of CD33 after treatment with mIgG1control antibody (FIG. 7H).

As shown in FIG. 7K-7N, treatment with the anti-Siglec-9 antibody 2D4was able to decrease cell surface levels of Siglec-9 in cells ofperipheral blood of the treated hu-NSG mice, when compared to isotypecontrol MOPC21 antibody treatment. Siglec-9 expression was decreased by˜80% as early as 1 day post antibody treatment with antibody 2D4 (FIG.7L). This decrease in Siglec-9 expression was sustained for 21 days posttreatment (FIGS. 7K and 7L). As a comparison, cell surface expression ofthe unrelated surface receptor CD33 did not decrease significantly afteranti-Siglec-9 antibody treatment, as compared to cell surface levels ofCD33 after treatment with mIgG1 control antibody (FIGS. 7K and 7M).

These results demonstrate that when utilizing humanized mice, Siglec-9antibodies engage the Siglec-9 in vivo and functionally downregulateSiglec-9 on human immune cells. The results further demonstrate thatSiglec-9 antibody 2D4, when injected into humanized mice, engageSiglec-9 on peripheral hCD45⁺ hCD14⁺ myeloid cells circulating in theblood of the mice. Antibody engagement of Siglec-9 leads to receptordownregulation specifically for Siglec-9 and not a control receptor,CD33. After a single injection of 2D4 antibody, 80% of Siglec-9expressed on human monocytes is downregulated 1 day after treatment.Receptor loss is sustained for 21 days after treatment with a singletreatment of antibody 2D4.

Effect of Siglec-9 Antibody on Expression of TREM2

To test the ability of Siglec-9 antibodies that decrease cell surfacelevels of Siglec-9 to modulate TREM2 expression, human monocyte-deriveddendritic cells differentiated with GM-CSF and IL-4 were harvested atday 6, and were plated at 500,000 cells per well in a 24 well dishovernight with 1.0 μg/ml anti-Siglec-9 antibody 2D4, isotype controlantibody, or left untreated. Cell surface receptor expression for CD11c,TREM2, and Siglec-9 was detected the following day by FACS analysis.Cells were incubated with biotinylated anti-TREM2 antibody 10A9 for 20minutes on ice. After one wash, cells were incubated in FACS buffer withAPC conjugated Streptavidin (BD Biosciences, 1:150 dilution),anti-Siglec-9-PE, clone K8 (Biolegend), and anti-CD11c-PECy7 (BDBiosciences), for 30 minutes on ice in the dark. Cells were washed 3× inFACS buffer (PBS+2% FBS, 2 mM EDTA) and flow cytometry was performed ona BD FACS Canto. Data was analyzed using TreeStar FlowJo software. Datawas calculated as a percent of receptor expression in the absence ofantibody using MFI values for the respective fluorophores.

As shown in FIGS. 7O and 7P, anti-Siglec-9 antibody 2D4, which was ableto able to decrease cell surface levels of Siglec-9, did not affectTREM2 expression.

Loss-of-function mutations in TREM2 have been associated with severeneurodegeneration, including Nasu-Hakola disease, frontotemporaldementia, and Alzheimer's disease. It is believed that modulation ofITIM-containing Siglec receptors may affect TREM2 expression. Therefore,anti-Siglec-9 antibodies that do not decrease TREM2 expression on humanimmune cells may be key to positive therapeutic outcomes. The results inFIGS. 70 and 7P demonstrate that anti-Siglec-9 antibody clone 2D4, whichrobustly decreases cell surface levels Siglec-9, does not reduce TREM2expression on human dendritic cells.

Example 4: Determining Whether Siglec-9 Antibodies Compete with Siglec-9Ligand for Binding to Human Siglec-9

The purpose of the following Example was to test whether anti-Siglec-9antibodies recognize the ligand-binding site on Siglec-9 and competewith ligand binding on Siglec-9 receptors on cells.

To determine whether Siglec-9 antibodies block binding to sialic acidligands, U937 cells, which robustly express sialic acid ligands thatpredominantly bind to Siglec-9, were utilized in a FACS-based protocol.Briefly, 25 μg/ml Siglec-9 antibodies were pre-coupled with 25 μg/mlSiglec-9-Fc (R&D Systems, catalog number 1139-SL-050) for 20 minutes onice in binding buffer (PBS with 0.25% BSA and 1 mM CaCl₂) in 96-wellround bottom plates. The U937 cells that were pre-blocked withanti-human CD64 antibody (10 μg/ml, Affymetrix catalog number 16-0649-85clone 10.1) were added to each well, mixed, and incubated on ice for 45minutes. Cells were pelleted and resuspended in goat anti-human IgG-PE(SouthernBiotech catalog number 2040-09) secondary antibody at 1:2000dilution in binding buffer for 30 minutes. Cells were then washed threetimes and analyzed by flow cytometry on a BD FACS Canto. Data wereanalyzed on FlowJo version 10.0.6 (TreeStar).

The results of the ligand competition assay are depicted in Table 10. InTable 10, “Siglec-9-Fc” refers to cells that were treated only withSiglec-9-Fc; “Isotype” refers to an isotype control antibody; and“Secondary Ab” refers to cells that were treated only with thePE-conjugated secondary antibody.

TABLE 10 Siglec-9 antibodies do not block Siglec-9-Fc binding to U937cells Antibody Percent Siglec-9-Fc Binding 2D4 99.2 2D5 97.8 5B1 97.86B2 97.8 6D8 97.8 7H12 97.8 Siglec-9-Fc 100 Isotype 0.021 Secondary Ab 0

As shown in Table 10, the Siglec-9 antibodies were not able to blockSiglec-9-Fc binding to U937 cells, thus indicating that the antibodiesdo not compete for binding to the ligand-binding site on Siglec-9, anddo not inhibit the interaction between Siglec-9 and one or more Siglec-9ligands (i.e., do not block ligand binding to Siglec-9).

In a separate experiment, U937 cells were cultured and passaged a fewtimes before experimental analysis. U937 cells were harvested withtrypsin or EDTA and plated ˜100,000 per well in U-Bottom 96-well plate.Cells were washed 1× in binding buffer (PBS+0.25% BSA and 1 mM CaCl₂),cells were resuspended in 100 μl binding buffer. In a separate 96-wellplate, Siglec-9-Fc was pre-incubated with anti-Siglec-9 antibody 2D4,5C6, 12B12, 17C2, or isotype control. Siglec-9-Fc fusion protein wasstored at a stock concentration of 50 μg/ml, rhIgG1-Fc serves asnegative control, and used 10 μl stock/well for 0.5 μg/well. Siglec-9antibodies were stored at 4° C. in 1.0 mg/ml stocks in PBS, and used 1μl/well for 1.0 μg/well total antibody. Fc fusion+antibody were added tosame well, allowed to bind on ice 10 minutes. Fc+/−mab was transferredto U937 cells, incubated on ice 30 minutes. Cells were washed 1× with100 ul Binding Buffer, pelleted at 1200 rpm for 5 minutes. Cells wereresuspended in 100 μl/well Binding buffer with Secondary Mouseanti-Human 1:2000 PE, and incubated with secondary on ice 30 minutes,washed 2× with binding buffer. FACS analysis for PE signal detection wasperformed on a FACSCanto (BD) and data analyzed with FlowJo (TreeStar)to compared MFI.

As shown in FIG. 7Q, Siglec-9 antibodies 5C6, 12B12, and 17C2 were ableto block receptor binding to endogenous sialic acid ligands expressed byU937 cells. Consistent with the results in Table 10, antibody 2D4 wasnot able to block ligand binding, and instead appeared to enhance theligand-receptor interaction (FIG. 7Q). The results indicate that ligandblocking may be one approach to prevent ITIM signaling and inhibitSiglec-9 receptor function.

Example 5: Summary of Siglec-9 Antibody Functional Studies

Table 11 summarizes results of the cell surface expression and ligandbinding studies described in Examples 3 and 4 above. As indicated inTable 11, there were three general classes of Siglec-9 antibodies. Oneclass of antibodies decreases cell surface level of Siglec-9 withoutinhibiting the interaction between Siglec-9 and one or more Siglec-9ligands. Antibodies in this class of antibodies include: 2D4, 5B1, 6B2,6D8, 7H12.

The second class of antibodies does not decrease cell surface level ofSiglec-9 and does not inhibit the interaction between Siglec-9 and oneor more Siglec-9 ligands. Antibodies in this class of antibodies include2D5.

The third class of antibodies decreases cell surface level of Siglec-9and inhibits the interaction between Siglec-9 and one or more Siglec-9ligands. Antibodies in this class of antibodies include: 5C6, 12B12, and17C2.

TABLE 11 Siglec-9 antibody functional studies Reduces Siglec-9 CellBlocks Ligand Antibody Surface Expression Binding 2D4 X 2D5 5B1 X 6B2 X6D8 X 7H12 X 5C6 X X 12B12 X X 17C2 X X

Example 6: Ligand Binding to Siglec-9 on Dendritic Cells Inhibits T CellProliferation and Phagocytosis

Human dendritic cells (DCs) were differentiated from peripheral bloodmonocytes with GM-CSF and IL-4 and cultured for 5 days. Immature(suspension) DCs were harvested and plated at a density of 200,000 cellsper ml in a 12 well dish. DCs were activated with a cytokine cocktail ofTNFa (50 μg/ml), IL-1b (50 μg/ml), IL-6 (150 ng/ml), and ProstaglandinE2 (1 μg/ml) for 24 hours. Dendritic cell maturation was determined byflow cytometry with commercially available antibodies for LIN, CD11c,HLA-DR, CD86, and CD83 (BD Biosciences). Immediately prior to co-culturewith allogenic isolated T cells, activated DCs were sialidase treated,or left untreated, for 2 hours at 37C with 100 mU/ml neuraminidase fromVibrio cholera in serum free media. Enzymatic activity was quenched byaddition of serum-containing media, cells pelleted and resuspended incomplete media. Sialidased activated, untreated activated, orunactivated DCs were co-cultured at a ratio of 1:10 with allogenic CFSElabeled T cells. CD3/CD28 Dynal beads were added to T cells alone as apositive control. Five days later T cell proliferation was measured byCFSE dilution on a BD FACS Canto.

FIGS. 8 and 9 show that sialic acids on dendritic cells restrict T cellproliferation during mixed lymphocyte reaction (MLR). FIG. 9 shows thatDCs that normally express inhibitory ligands induce low levels of T cellproliferation (left panel), while the removal of the inhibitory ligandson DCs increases T cell proliferation (right panel).

As shown in FIGS. 8 and 9, enzymatic removal of sialic acids fromactivated DCs increased T cell proliferation when compared to untreatedactivated DCs. These results indicate that sialic acids present on DCsact on T cells in a suppressive manner to restrict T cell proliferationwhen co-cultured with allogenic DCs. These results indicate thatantibodies that block Siglec-9 on T cells or dendritic cells enhance Tcell and/or dendritic cell functionality. CD3/CD28 Dynal beads were usedas a positive control. Furthermore, these results indicate that blockingsialic acid interactions with DCs or any other cellular or biologicalsource may increase T cell function.

Example 7: Inflammatory Conditions Induce Siglec-9 Sialic Acid LigandExpression in Myeloid Cells

Human dendritic cells (DCs) were differentiated from peripheral bloodmonocytes with GM-CSF and IL-4 and cultured for 5 days. Immature humanDCs were harvested on day 5 and co-cultured with sterile-filteredsupernatant from B 16, Lewis lung, MC38 tumor supernatant or 10 ng/mlLPS. 24 hours later Siglec-9 expression was determined by flow cytometrywith a directly conjugated Siglec-9-PE antibody. Sialic acid ligandexpression was assessed by incubation for 30 minutes on ice with 50μg/ml soluble Siglec-9 fused to human IgG1-Fc, IgG1-Fc alone was used anegative control in the presence of human Fc block. Binding of thesoluble receptor to sialic acids on cells was detected after a wash stepand incubation for 30 minutes on ice with anti-human secondaryconjugated to PE. Flow cytometry analysis was performed on a BD FACSCanto.

To elicit primary macrophages, human monocytes from peripheral humanblood samples are isolated and either used directly or differentiatedinto macrophages with 50 μg/ml M-CSF for 5 days. In order to determinethe role of Siglec-9 in inflammatory cytokine production, humanmacrophages are cultured with various inflammatory mediators, andcytokine levels are measured in the culture supernatants. To generatehuman macrophages, monocytes from peripheral human blood samples areisolated and either used directly or differentiated into macrophageswith 50 μg/nl M-CSF or dendritic cells with 100 μg/ml GM-CSF and 100μg/ml IL-4 for 5 days. Cells are cultured for 5 days, and adherent cellswere detached with 1 mM EDTA in PBS. Cells are plated on 96-well platesat 10⁵ cells/well and allowed to adhere for 4 h at 37° C. Cells are thenstimulated with TLR agonists LPS (Salmonella abortus equi) or zymosan(Saccharomyces cerevisiae) at concentrations ranging from 0.01-100 ng/ml(LPS) or 0.01-100 μg/ml (zymosan). Alternatively, macrophages arecultured in the presence of 10 ng/ml of the cytokine IL-4 or 50 ng/ml ofIFN-γ. Cell culture supernatant are collected 24 or 48 hours afterstimulation and the levels of TNFa, IL-6, IL-10, and MCP-1 cytokines aremeasured by using Cytometric Bead Array Inflammation Kit (BD) accordingto manufacturer's protocol. Macrophages stimulated with the inflammatorymediators LPS or zymosan are expected to secrete more inflammatorycytokines TNFa, IL-6, IL-10, and MCP-1 when treated with Siglec-9antagonistic antibodies or with enzymes that remove the inhibitoryglycol ligands.

FIGS. 10A and 10B show that Siglec-9 expression is maintained on humandendritic cells after exposure to tumor supernatant. FIGS. 10C and 10Dshow that tumor supernatant increases expression of sialic acid (aSiglec-9 ligand). FIGS. 10E and 10F show that Siglec-9 expression ismaintained on human dendritic cells during LPS-induced inflammation.FIGS. 10G and 10H show that LPS-induced inflammation increasesexpression of sialic acid (a Siglec-9 ligand).

These results indicate that inflammatory conditions and tumorenvironment lead to upregulation of both Siglec-9 and sialic acidligands. The results also demonstrate that increased Siglec-9 functioncan immunosuppress human primary dendritic cells. These results indicatethat inhibiting Siglec-9 with downregulating or blocking antibodies mayrelieve immunosuppressed myeloid-derived or tumor-associated myeloidcells and restore immune function. These results further indicate thatantibodies that block Siglec-9 on myeloid cells enhance myeloid cellfunctionality.

Example 8: Increased E. coli Phagocytosis by Dendritic Cells withSialidase Treatment

The purpose of the following Example was to test whether antagonisticanti-Siglec-9 antibodies and/or Siglec-9 bispecific antibodies inducephagocytosis of apoptotic neurons, nerve tissue debris, non-nerve tissuedebris, bacteria, other foreign bodies, and disease-causing proteins,such as A beta peptide, alpha synuclain protein, Tau protein, TDP-43protein, prion protein, huntingtin protein, RAN, translation productsantigene, including the DiPeptide Repeats, (DPRs peptides) composed ofglycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR),proline-alanine (PA), or proline-arginine (PR) in cells from the myeloidlineage, such as monocytes, dendritic cells macrophages and microglia.The bispecific antibodies may be antibodies that recognize the Siglec-9antigen and a second antigen that includes, without limitation, CD3, Abeta peptide, antigen or an alpha synuclain protein antigene or, Tauprotein antigene or, TDP-43 protein antigene or, prion protein antigeneor, huntingtin protein antigene, or RAN, translation Products antigene,including the DiPeptide Repeats, (DPRs peptides) composed ofglycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR),proline-alanine (PA), or proline-arginine (PR).

Monocytes from peripheral human blood samples were isolated using theRosetteSep™ monocyte isolation antibody cocktail (StemCellTechnologies), and differentiated into dendritic cells with GM-CSF andIL-4 (PeproTech) and cultured for 5 days. Cells were plated on culturedishes in RPMI medium (Invitrogen) containing 10% fetal calf serum(Hyclone) and cultured at 37° C. in 5% CO₂. Non-adherent cells werecollected and used for phagocytosis experiments.

To conduct bacterial phagocytosis assay, dendritic cells were harvestedand plated in 96 well flat bottom plates without cytokine for 2 hours.pHrodo-labeled E. coli BioParticles were resuspended according tomanufacturer's protocol and were treated with 0.2 U/ml or 0.4 U/mlsialidase from Vibro cholera, or PBS alone for 2.5 hours at 37° C.BioParticles were washed, resuspended in RPMI and added 20 ug/well.Dendritic cells and E. coli cells were mixed, pelleted, and incubated at37° C. for 30 minutes. Cytochalasin D was added at 10 uM to controlwells. Immediately prior to FACS analysis, cells were transferred to iceand washed 2× in FACS buffer at 4° C. pHrodo-labeled E. coliphagocytosis was detected in the PE channel by flow cytometry on a BDFACS Canto.

FIG. 11 shows that sialidase treatment increased dendritic cell-mediatedphagocytosis of E. coli. These results indicate that antagonisticanti-Siglec-9 antibodies and/or Siglec-9 bispecific antibodies that, forexample, decrease cell surface expression of Siglec-9 and/or inhibit thebinding of one or more Siglec-9 ligands to Siglec-9 can also be used toinduce or otherwise increase phagocytosis.

Example 9: Increased Expression of Siglec-9 Ligand in Brain Sections ofAlzheimer's Disease Patients

Sialic acid ligand expression was detected in the brains of normal andAlzheimer's disease (AD) patients by immunohistochemistry withbiotinylated Siglec-9-Fc (R&D). The Siglec-9-Fc protein was biotinylatedwith the EZ-Link Sulfo-NHS-Biotin (Thermo Scientific) according tomanufacturer's instructions. The IHC procedure, with the exception ofthe overnight incubation, was performed on a shaker. Samples wereincubated for 15 minutes in 10% MeOH, 3% H₂O₂ in PBS, followed by 3washes in PBS with 4% serum. Next, samples were incubated for 30 minutesin 0.2% Triton™-X100, 4% serum, 0.019% L-lysine in PBS, followed by anhour in primary antibody then overnight at 4° C. in PBS with 4% serum.The next day samples were placed on a shaker for one hour followed by 3washes, then samples were incubated for one hour in ABC buffer andwashed 3 times. Samples were developed with a Vector DAB peroxidase kit,washed 3 times and dehydrated and imaged with a Nikon 90i microscopewith color camera, magnification of 200×. The quantification wasperformed using Nikon Elements BR image analysis software.

FIG. 12 shows that sialic acid Siglec-9 ligands are upregulated in brainsections from two AD patients (Donor 1 and Donor 2). Data from 5 AD and5 non-AD human brains show a statistically significant increase inexpression of Siglec-9 sialic acid ligands by one way ANOVA, p=0.0159(FIG. 12).

These results indicate that antibodies that remove Siglec-9 from thecell surface or block increased ligand interactions may relieveinhibitory Siglec-9-dependent signaling on microglia or other myeloidcells in the brain and restore normal functions to these cells, withbeneficial effects for Alzheimer's disease.

Example 10: Increased Expression of Siglec-9 Ligand in Cancer Cells

In Vitro Studies

Mouse melanoma (B16), Lewis lung tumor cells, or colon carcinoma (MC38)were cultured and incubated with 50 μg/ml Siglec-9-Fc or an IgG1-Fccontrol on ice for 30 minutes to determine the level of Siglec-9 bindingsialic acid ligand expression on these cell types. Binding interactionswere performed in PBS with 0.25% BSA, 1 mM CaCl₂. To detect Siglec-9receptor binding, a goat anti-human Fc-PE secondary antibody wasincubated for 30 minutes on ice after washing off unbound Siglec-9-Fc.Cells were washed three times in Binding Buffer and analyzed by flowcytometry on a BD FACS Canto.

FIG. 13 shows that the expression of an inhibitory Siglec-9 ligand isincreased at least 20-fold over background in melanoma cells, lung tumorcells, and colon cancer cells. Without wishing to be bound by theory,identification of inhibitory sialic acid ligand expression on thesetumor cells indicates a contributing mechanism by which cancer cellsevade immune recognition and clearance. Sialic acid ligands on tumorcells can mediate immunosuppressive interactions via Siglec-9 expressedon myeloid and lymphoid immune cells. These results indicate thatantibodies that remove Siglec-9 from the cell surface or block increasedligand interactions may relieve inhibitory effects of tumors on theimmune system and enhance cancer therapy.

In Vivo Studies

Four week-old female Taconic NOG mice were myeloablated approximately 24hours before engraftment with human fetal liver CD34⁺ cells (100,000cells/mouse) by intravenous injection. Reconstitution of immune cellswas monitored by flow cytometry of peripheral blood. Twelve weeks afterengraftment, the Champions tumorgraft melanoma model was implantedsubcutaneously. Approximately 8-10 weeks later, when the tumor reached asize of 150-200 mm³, blood, spleen and tumors were harvested andprocessed for analysis by flow cytometry on a BD FACS Canto. Flowcytometric analysis was performed to determine the expression ofSiglec-9 in different compartments of the human (hCD45⁺) immune system.Specifically, expression was analyzed in CD3⁺ T cells, CD14⁺monocyte/macrophages and other CD3−CD14− human immune cells. Data wereanalyzed with FlowJo software version 10.0.6 by TreeStar.

As depicted in FIG. 14, the results indicated that Siglec-9 is expressedin peripheral blood, spleen, and tumor infiltrating immune cells intransplanted melanoma tumors of humanized mice. Importantly, Siglec-9 issignificantly upregulated in human immune cells infiltrating melanomatumors. This demonstrates the relevance of this mouse model to assessingthe therapeutic ability of Siglec-9 antibodies.

Example 11: Reduction of the Anti-Inflammatory Cytokine IL-10 in MyeloidCells by Antagonistic and/or Bispecific Siglec-9 Antibodies

The purpose of this Example is to test whether bone marrow-derivedmyeloid cells show a decrease in the anti-inflammatory cytokine IL-10and other anti-inflammatory mediators following treatment withantagonistic anti-Siglec-9 and/or Siglec-9 bispecific antibodies andstimulation with 100 ng/ml LPS (Sigma), by co-culturing with apoptoticcells, or by a similar stimulus.

Isolation of human myeloid precursor cells is performed as previouslydescribed. Medium is changed after 5 d and cells are cultured for anadditional 10-11 d. Supernatant is collected after 24 h, and the levelof IL-10 and other anti-inflammatory cytokines released from the cellsis determined by IL-10 ELISA according to manufacturer's instructions(R&D Systems) (JEM (2005), 201; 647-657; and PLoS Medicine (2004),4|Issue 4|e124).

Example 12: Induction of Phagocytosis in Cells from the Myeloid Lineageby Antagonistic and/or Bispecific Siglec-9 Antibodies

The purpose of this Example is to test whether antagonisticanti-Siglec-9 antibodies and/or Siglec-9 bispecific antibodies inducephagocytosis of apoptotic neurons, nerve tissue debris, non-nerve tissuedebris, bacteria, other foreign bodies, and disease-causing proteins,such as A beta peptide, alpha synuclain protein, Tau protein, TDP-43protein, prion protein, huntingtin protein, RAN, translation productsantigene, including the DiPeptide Repeats, (DPRs peptides) composed ofglycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR),proline-alanine (PA), or proline-arginine (PR) in cells from the myeloidlineage, such as monocytes, Dendritic cells macrophages and microglia.The bispecific antibodies may be antibodies that recognize the Siglec-9antigen and a second antigen that includes, without limitation, A betapeptide, antigen or an alpha synuclain protein antigene or, Tau proteinantigene or, TDP-43 protein antigene or, prion protein antigene or,huntingtin protein antigene, or RAN, translation Products antigene,including the DiPeptide Repeats, (DPRs peptides) composed ofglycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR),proline-alanine (PA), or proline-arginine (PR).

Monocytes from peripheral human blood samples are isolated using theRosetteSep monocyte isolation antibody cocktail (StemCell Technologies)and differentiated into macrophages, neutrophils, and NK cells with 50μg/ml M-CSF (PeproTech) for 5 days. Cells are plated on culture dishesin RPMI medium (Invitrogen) containing 10% fetal calf serum (Hyclone)and cultured at 37° C. in 5% CO₂. Adherent cells are collected by gentlescraping and used for phagocytosis experiments.

Human microglial cells are prepared from peripheral blood monocytes byculture in serum-free RPMI with 1% Pen/Strep, 10 ng/ml GM-CSF, 10 ng/mlM-CSF, 10 ng/ml beta-NGF, 100 ng/ml CCL-2, 100 ng/ml IL-34 according toprotocols described in Etemad et al., JI (2012), and Ohgidani et al.,Scientific Reports (2014). Cells were harvested at day 7-10 whenramified morphology appeared.

To conduct phagocytosis assays microglia, macrophages, neutrophils, NKcells or dendritic cells are cultured with apoptotic neurons, nervetissue debris, non-nerve tissue debris, bacteria, other foreign bodies,and disease-causing proteins. Neurons are cultured for 5-10 d, andokadaic acid is then added at the final concentration of 30 nM for 3 hto induce apoptosis. Neuronal cell membranes are labeled withCellTracker CM-DiI membrane dye (Molecular Probes). After incubation,apoptotic neurons or other targets of phagocytosis are washed two timesand added to the transduced microglial culture at an effector/targetratio of 1:20. At 1 and 24 h after addition of apoptotic neurons, thenumber of microglia having phagocytosed neuronal cell membranes iscounted under a confocal fluorescence microscope (Leica). Apoptoticcells are counted in three different areas at a magnification of 60. Theamount of phagocytosis is confirmed by flow cytometry. Moreover, 24, 48,or 72 h after the addition of apoptotic neurons, cells are collected andused for RT-PCR of cytokines.

To conduct microsphere bead or bacterial phagocytosis assay, microglia,macrophages, neutrophils, NK cells, or dendritic cells are treated withanti-Siglec-9 agonistic antibodies. Cells are harvested and plated in 96well flat bottom plates without cytokine for 2 hours. pHrodo-labeled E.coli BioParticles are resuspended according to manufacturer's protocoland are treated with 0.2 U/ml or 0.4 U/ml sialidase from Vibro cholera,or PBS alone for 2.5 hours at 37° C. BioParticles are washed,resuspended in RPMI and added 20 μg/well. Cells and E. coli were mixed,pelleted, and incubated at 37° C. for 30 minutes. Cytochalasin D isadded at 10 uM to control wells. Immediately prior to FACS analysis,cells are transferred to ice and washed 2× in FACS buffer at 4° C. ThepHrodo-labeled E. coli phagocytosis is detected in the PE channel byflow cytometry on a BD FACS Canto.

After 24 h, 1.00 μm of red fluorescent microsphere heads (FluoresbritePolychromatic Red Microspheres; Polysciences Inc.) are added for 1 h.Phagocytosis of microsphere beads by microglia is analyzed byfluorescence microscopy. Furthermore, microglia are collected from theculture plates and analyzed by flow cytometry. The percentage ofmicroglia having phagocytosed beads is determined. Because phagocytosisvaries from one experiment to the other, the relative change inphagocytosis is also determined. Data are shown as the relative changein phagocytosis between microglia cultured with agonistic antibodies andcontrol antibody.

To conduct RT-PCR for analysis of inflammatory gene transcripts,microglia are transduced with a Siglec-9 vector or a GFP1 controlvector. Cells are then cultured on dishes and treated with anti-Siglec-9agonistic antibodies. After 24, 48, and 72 h, RNA is isolated frommicroglia using an RNeasy Mini Kit (QIAGEN). RNA is also collected frommicroglia that have been transduced with sh-Siglec-9 RNA, sh-controlRNA, wSiglec-9, GFP2, mtDAP12-GFP, and GFP1 vector and co-cultured withapoptotic neurons for 48 h.

Reverse transcription of RNA is then performed. Quantitative RT-PCR bySYBR Green is performed on an ABI Prism 5700 Sequence Detection System(PerkinElmer). Amplification of GAPDH is used for sample normalization.The amplification protocol followed the GeneAmp 5700 Sequence DetectionSystem Software (version 1.3). For detection of GAPDH, TNF-alpha, IL-1,NOS2, and TGF-beta transcripts, the following forward and reverseprimers were used at final concentrations of 200 nM:

GAPDH forward primer: (SEQ ID NO: 241) 5′-CTCCACTCACGGCAAATTCAA-3′, andGAPDH reverse primer: (SEQ ID NO: 242) 5′-GATGACAAGCTTCCCATTCTCG-3′;TNF-α forward primer: (SEQ ID NO: 243) 5′-CCGTCAGCCGATTTGCTATCT-3′, andTNF-α reverse primer: (SEQ ID NO: 244) 5′-ACGGCAGAGAGGAGGTTGACTT-3′;IL-1α forward primer: (SEQ ID NO: 245) 5′-ACAA-CAAAAAAGCCTCGTGCTG-3′,and IL-1α reverse primer: (SEQ ID NO: 246) 5′-CCATTGAGGTGGAGAGCTTTCA-3′;NOS2 forward primer: (SEQ ID NO: 247) 5′-GGCAAACCCAAGGTCTACGTTC-3′,NOS2 reverse primer: (SEQ ID NO: 248) 5′-TACCTCATTGGCCAGCTGCTT-3′; andTGF-β1 forward primer: (SEQ ID NO: 249) 5′-AGGACCTGGGTTGGAAGTGG-3′, andTGF-β1 reverse primer: (SEQ ID NO: 250) 5′-AGTTGGCATGGTAGCCCTTG-3′.

To conduct amyloid phagocytosis assay, HiLyteFluor™ 647(Anaspec)-Abeta-(1-40) is resuspended in Tris/EDTA (pH 8.2) at 20 mM andthen incubated in the dark for 3 d at 37° C. to promote aggregation.Microglia, macrophages, neutrophils, NK cells, or dendritic cells arepretreated in low serum (0.5% FBS supplemented with insulin), LPS (50ng/ml), IFNc (100 units/ml), and anti-Siglec-9 antagonistic antibodiesfor 24 h prior to the addition of aggregated fluorescently labeled abeta peptide. Amyloid phagocytosis and surface expression of Siglec-9are determined by flow cytometric analysis 5 h post-addition of 100 nMaggregated HiLyteFluor™ 647-Ab-(1-40) (ASN NEURO (2010) 2(3): 157-170).Phagocytosis of other disease-causing proteins is conducted in a similarmanner.

Example 13: Induction of SYK and/or ERK Activation by AntagonisticSiglec-9 Antibodies and/or Bispecific Antibodies

The purpose of this Example is to test whether agonistic anti-Siglec-9antibodies and/or Siglec-9 bispecific antibodies induce Syk and ERKactivation.

Microglia, macrophages, neutrophils, NK cells, or dendritic cells areexposed to agonistic anti-Siglec-9 and/or Siglec-9 bispecific antibodiesfor 1 h. After stimulation, cells are lysed in reducing sample bufferfor Western blot analysis. Phosphorylation of ERK and total amount ofSyk and/or ERK are determined by immuno-detection with anti-phospho-Sykor ERK and anti-Syk or ERK antibodies, respectively (both from CellSignaling Technology) by Western blot analysis (JEM (2005), 201,647-657).

Example 14: Siglec-9 Antibodies and/or Bispecific Antibodies Induce SykPhosphorylation

Spleen tyrosine kinase (Syk) is an intracellular signaling molecule thatfunctions downstream of Siglec-9 by phosphorylating several substrates,thereby facilitating the formation of a signaling complex leading tocellular activation and inflammatory processes. The ability of agonistSiglec-9 antibodies to induce Syk activation is determined by culturinghuman macrophages, human neutrophils, human NK cells, and human primarydendritic cells and measuring the phosphorylation state of Syk proteinin cell extracts.

Human primary dendritic cells are starved for 4 hours in 1% serum RPMIand then removed from tissue culture dishes with PBS-EDTA, washed withPBS, and counted. The cells are coated with full-length agonist Siglec-9antibodies, or control antibodies for 15 minutes on ice. After washingwith cold PBS, cells are incubated at 37° C. for the indicated period oftime in the presence of goat anti-human IgG. After stimulation, cellsare lysed with lysis buffer (1% v/v NP-40%, 50 Mm Tris-HCl (pH 8.0), 150mM NaCl, 1 mM EDTA, 1.5 mM MgCl₂, 10% glycerol, plus protease andphosphatase inhibitors) followed by centrifugation at 16,000 g for 10min at 4° C. to remove insoluble materials. Lysates are thenimmunoprecipitated with anti-Syk Ab (4D10 for human DCs, Santa CruzBiotechnology). Precipitated proteins are fractionated by SDS-PAGE,transferred to PVDF membranes and probed with anti-phosphotyrosine Ab(4G10, Millipore). To confirm that all substrates are adequatelyimmunoprecipitated, immunoblots are reprobed with anti-Syk (NovusBiological, for human DCs). Visualization is performed with the enhancedchemiluminescence (ECL) system (GE healthcare), as described (e.g., Penget al., (2010) Sci Signal., 3(122): ra38).

Example 15: Induction of CCR7 and Migration Toward CCL19 and CCL21 inMicroglia, Macrophages, Neutrophils, NK Cells, and Dendritic Cells byAntagonistic Siglec-9 Antibodies and/or Bispecific Antibodies

The purpose of this Example is to test whether anti-Siglec-9 antibodiesand/or Siglec-9 bispecific antibodies induce CCR7 and migration towardCCL19 and CCL21 in microglial cells, macrophages, neutrophils, NK cells,and dendritic cells.

Microglial, macrophages or dendritic cells are either cultured withagonistic anti-Siglec-9 and/or Siglec-9/DAP12 bispecific antibodies, orwith a control antibody. Cells are collected after 72 h, immuno-labeledwith CCR7 specific anti-bodies, and analyzed by flow cytometry.

To determine any functional consequences of increased CCR7 expression, achemotaxis assay is performed. Microglia, macrophages, neutrophils, NKcells, or dendritic cells are stimulated via Siglec-9 with theantagonistic anti-Siglec-9 and/or Siglec-9/DAP12 bispecific antibodiesand placed in a two-chamber system. The number of microglial cellsmigrating toward the chemokine ligands CCL19 and CCL21 is quantified(JEM (2005), 201, 647-657).

For the chemotaxis assay, microglial, macrophages, neutrophils, NKcells, or dendritic cells are exposed to the antagonistic anti-Siglec-9antibodies or Siglec-9 bispecific antibodies and treated with 1 μg/mlLPS. Microglia, macrophages, neutrophils, NK cells, or dendritic cellsare transferred into the upper chamber of a transwell system (3 m porefilter; Millipore) containing 450 μl medium with 100 ng/ml CCL19 orCCL21 (both from PeproTech) in the lower chamber. After a 1 h incubationperiod, the number of microglial macrophages, neutrophils, NK cells, ordendritic cells that have migrated to the lower chamber is counted inthree independent areas by microscopy (JEM (2005), 201, 647-657).

Example 16: Induction of F-Actin in Microglia, Macrophages, Neutrophils,NK Cells, and Dendritic Cells by Antagonistic Siglec-9 Antibodies and/orBispecific Antibodies

The purpose of this Example is to test whether antagonisticanti-Siglec-9 antibodies, or Siglec-9 bispecific antibodies induceF-actin in microglial cells, macrophages, neutrophils, NK cells, anddendritic cells.

Microglia, macrophages, neutrophils, NK cells, or dendritic cells andother cells of interest that are transduced with Siglec-9 or thatexpress Siglec-9 are added to culture plates and then exposed toantagonistic anti-Siglec-9 and/or Siglec-9 bispecific antibodies, or acontrol antibody. Cells are fixed, blocked, and then stained with AlexaFluor 546-conjugated phalloidin (Molecular Probes) after 1 h and F-actinis labeled with a fluorescence dye. Images are collected by confocallaser scanning microscopy with a 40× objective lens (Leica). (JEM(2005), 201, 647-657).

Example 17: Induction of Osteoclast Production and Increased Rate ofOsteoclastogenesis by Antagonistic Siglec-9 Antibodies and/or BispecificAntibodies

The purpose of this Example is to test whether antagonisticanti-Siglec-9 antibodies and/or Siglec-9 bispecific antibodies induceosteoclast production and increase the rate of osteoclastogenesis.

Human monocyte derived monocyte/macrophage are maintained in RPMI-1640medium (Mediatech), or another appropriate medium, supplemented with 10%FBS (Atlantic Biologics, Atlanta, Ga., USA) andpenicillin-streptomycin-glutamine (Mediatech). Cells are seeded in96-well plates with 3000 cells/well in alpha-MEM medium supplementedwith 10% FBS, penicillin-streptomycin-glutamine, 50 ng/ml RANKL, and 20ng/ml M-CSF. The medium is changed every 3 days, exposed toanti-Siglec-9 antagonistic antibodies and the number of multinucleated(at least three nuclei) TRACP⁺ osteoclasts are counted and scored bylight microscopy. To determine complexity and size, osteoclasts arecounted by number of nuclei (>10 or 3-10 nuclei). The surface area ofosteoclasts is also measured by using Image J software (NIH). Inaddition, expression levels of osteoclasts genes are determined. TotalRNA is extracted from osteoclastogenic cultures at different time pointsusing TRIzol reagent (Invitrogen). After first-strand cDNA synthesisusing a SuperScript III kit (Invitrogen), real-time quantitative PCRreactions are performed for Nfatc1, Acp5, Ctsk, Calcr, and Ccnd1.Relative quantification of target mRNA expression is calculated andnormalized to the expression of cyclophilin and expressed as (mRNA ofthe target gene/mRNA of cyclophilin) 3×10⁶. (J. OF BONE AND MINERALRESEARCH (2006), 21, 237-245; J Immunol 2012; 188:2612-2621).

Alternatively, macrophages, neutrophils, or NK cells are seeded onto theplates in triplicate wells and treated with RANKL, M-CSF, and with ananti-Siglec-9 and/or Siglec-9 bispecific antibody, or an isotype-matchedcontrol monoclonal antibody. The medium is changed every 3 days untillarge multinucleated cells are visible. After 3 to 5 days in culture,cells are fixed with 3.7% formaldehyde in PBS for 10 min. Plates arethen washed twice in PBS, incubated for 30 s in a solution of 50%acetone and 50% ethanol, and washed with PBS. Cells are stained fortartrate-resistant acid phosphatase (TRAP) with a kit from Sigma(product 435). Multinucleated (more than two nuclei), TRAP-positivecells are then counted by light microscopy, as described (e.g., Peng etal., (2010) Sci Signal., 3(122): ra38).

Example 18: In Vivo Protection from EAE and Cuprizone in a Whole Animal

Adult 7-9 week-old female C57BL/6 mice (obtained from Charles RiverLaboratories) are injected in the tail base bilaterally with 200 μl ofan innoculum containing 100 μg of myelin oligodendrocyte glycoproteinpeptide 35-55 (amino acids MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 251);Seqlab) and 1 mg of Mycobacterium tuberculosis H37 Ra (Difco) inincomplete Freund adjuvant (Difco). Pertussis toxin (200 ng; ListBio-logical Laboratories) is injected at day 0 and at day 2 afterimmunization. Clinical signs are scored as follows: 0, no clinicalsigns; 1, complete limp tail; 2, complete limp tail and abnormal gait;3, one hind-limb paraparesis; 4, complete hindlimb paraparesis; and 5,fore- and hind-limb paralysis or moribund. Only mice having diseaseonset (clinical score of 1 or more) at day 14 are used for experiments.Agonistic anti-Siglec-9 and/or Siglec-9 bispecific antibodies areinjected intraperitoneally or intravenously in EAE-diseased mice at theday of the first clinical symptoms or at any other desired time (PLoSMed (2007) 4(4): e124).

Young or aged wild-type (WT) mice are fed a standard diet (Harlan)containing 0.2% cuprizone (CPZ) powdered oxalicbis(cyclohexylidenehydrazide) (Sigma-Aldrich) for 4, 6 or 12 weeks. ForHistological and immunohistochemical analyses brains are removed aftermouse perfusion with 4% paraformaldehyde (PFA), fixed in 4% PFA for 24h, followed by immersion in 30% sucrose for 24-48 h. To evaluate myelinintegrity and damage, as well as cell proliferation and inflammationsections or mouse brain are stained with anti-MBP (1:100; Abcam,ab7349), -dMBP (1:2000; Millipore, ab5864), -β APP (1:100; Invitrogen,51-2700), -SMI-31 (1:1000; Covance, smi-31R), -Iba1 (1:600; Wako,019-19741), -BrdU (1:250; Abcam, ab1893), -GFAP (1:200; Invitrogen,13-0300), -iNOS (1:100; BD Pharmingen, 610329), -LPL(1:400, from Dr. G.Olivecrona) and -MHC II (1:100; BD Pharmingen, 553549). For behavioraleffects of the antibodies, mice are analyzed for locomotor activityusing transparent polystyrene enclosures and computerized photobeaminstrumentation. General activity variables (total ambulations, verticalrearings), along with indices of emotionality including time spent,distance traveled and entries, are analyzed. A battery of sensorimotortests is performed to assess balance (ledge and platform), strength(inverted screen), coordination (pole and inclined screens) andinitiation of movement (walking initiation). Motor coordination andbalance are studied using a rotarod protocol (Cantoni et al., ActaNeuropathol (2015)129(3):429-47).

Example 19: Characterization of the Therapeutic Use of AntagonisticSiglec-9 Antibodies and/or Siglec-9 Bispecific Antibodies in EstablishedAnimal Models of Traumatic Brain Injury

The therapeutic utility of antagonistic anti-Siglec-9 and/or Siglec-9bispecific antibodies is tested in established animal models oftraumatic brain injury (Tanaka, Y et al. (2013) Neuroscience 231 49-60).Either regular mice or mice that express the human Siglec-9 gene under abacterial artificial chromosome or under a myeloid promoter can be used.For example, a model of traumatic brain injury that induces theactivation of microglia and astrocytes is used. Eight or nine week-oldmale C57BL/6J WT mice are used (purchased from Charles RiverLaboratories or Jackson Laboratories). Mice are anesthetized byintraperitoneal administration of xylazine hydrochloride (8 mg/kg) andchloral hydrate (300 mg/kg) dissolved in sterile saline, andsubsequently placed in a stereotaxic apparatus (Narishige, Tokyo,Japan). An incision is made in the scalp and the cranium is exposed. Theperiosteum is cleaned from the skull, a hole is drilled over the rightcerebral henmisphere with a dental drill, and the duramater is removedwith a needle tip. A stainless steel cannula, with a 0.5 mm outerdiameter, is used to make a longitudinal stab wound in the righthemisphere. The cannula is positioned at 1.3 mm lateral to the midline,and 1 mm posterior to bregma, and introduced into the brain until thetip reaches a depth of 2 mm. The cannula is then shifted 2 mm caudally(bregma 3 mm), and then shifts back 2 mm rostrally to its initialposition. Finally, the cannula is removed from the brain, and the scalpwound is sutured. Mice are then treated with antagonistic anti-Siglec-9and/or Siglec-9 bispecific antibodies according to standard proceduresand then analyzed by histology and immunofluorescence staining andbehavioral tests. Such experiment can be also conducted in mice thatexpress the human Siglec-9 gene from a bacterial artificial chromosomeor from a cDNA driven by a myeloid promoter or in mice that weretransduced with lenti or AAV virus containing hSiglec-9 cDNA.

Example 20: Characterization of Therapeutic Use of Antagonistic Siglec-9Antibodies and/or Siglec-9 Bispecific Antibodies in a Model ofNeuro-Inflammation and Neuron Loss Following Toxin-Induced Injury

The therapeutic utility of agonistic anti-Siglec-9 and/or Siglec-9bispecific antibodies is tested in a model of neuro-inflammation andneuron loss following toxin-induced injury (Martens, L H et al., (2012)The Journal of Clinical Investigation, 122, 3955). Three-month-oldregular mice, are treated with 4 intraperitoneal injections of MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) per day for 2 days (4μg/g body weight) (Sigma-Aldrich) or PBS. Mice are treated withagonistic anti-Siglec-9 and/or Siglec-9 bispecific antibodies accordingto standard protocols and then analyzed using Stereological counting toquantify dopamine neurons and microglia in the substantia nigra parscompacta (SNpc), as described. Such experiment can be also conducted inmice that express the human Siglec-9 gene from a bacterial artificialchromosome or from a cDNA driven by a myeloid promoter or in mice thatwere transduced with lenti or AAV virus containing hSiglec-9 cDNA.

Example 21: Characterization of the Therapeutic Use of AntagonisticSiglec-9 Antibodies and/or Siglec-9 Bispecific Antibodies in AnimalModels of Aging, Seizures, Spinal Cord Injury, Retinal Dystrophy,Frontotemporal Dementia, and Alzheimer's Disease

The therapeutic utility of antagonistic anti-Siglec-9 and/or Siglec-9bispecific antibodies is tested in animal models for aging, seizures,spinal cord injury, retinal dystrophy, frontotemporal dementia,Huntington disease, Parkinson's disease amyotrophic lateral sclerosisand Alzheimer's disease, as previously described (e.g., Beattie, M S etal., (2002) Neuron 36, 375-386; Volosin, M et al., (2006) J. Neurosci.26, 7756-7766; Nykjaer, A et al., (2005) Curr. Opin. Neurobiol. 15,49-57; Jansen, P et al., (2007) Nat. Neurosci. 10, 1449-1457; Volosin, Met al., (2008) J. Neurosci. 28, 9870-9879; Fahnestock, M et al., (2001)Mol. Cell Neurosci. 18, 210-220; Nakamura, K et al., (2007) Cell Death.Differ. 14, 1552-1554; Yune, T et al., (2007) Brain Res. 1183, 32-42;Wei, Y et al., (2007) Neurosci. Lett. 429, 169-174; Provenzano, M J etal., (2008) Laryngoscope 118, 87-93; Nykjaer, A et al., (2004) Nature427, 843-848; Harrington, A W et al., (2004) Proc. Natl. Acad. Sci.U.S.A. 101, 6226-6230; Teng, H K et al., (2005) J. Neurosci. 25,5455-5463; Jansen, P et al., (2007) Nat. Neurosci. 10, 1449-1457;Volosin, M et al., (2008) J. Neurosci. 28, 9870-9879; Fan, Y J et al.,(2008) Eur. J. Neurosci. 27, 2380-2390; Al-Shawi, R et al., (2008) Eur.J. Neurosci. 27, 2103-2114; and Yano, H et al., (2009) J. Neurosci. 29,14790-14802). Such experiment can be also conducted in mice that expressthe human Siglec-9 gene from a bacterial artificial chromosome or from acDNA driven by a myeloid promoter or in mice that were transduced withlenti or AAV virus containing hSiglec-9 cDNA.

Example 22: Characterization of the Therapeutic Use of AntagonisticSiglec-9 Antibodies and/or Siglec-9 Bispecific Antibodies in a Model ofInfection

The therapeutic utility of antagonistic anti-Siglec-9 antibodies and/orSiglec-9 bispecific antibodies is tested in a model of infection. Forexample, Listeria monocytogenes or other infection in normal mice can beused, as previously described (e.g., Yin, F et al., (2009) J. Exp. Med,207, 117-128). Either regular mice or mice that express the humanSiglec-9 gene under a bacterial artificial chromosome or under a myeloidpromoter can be used.

Example 23: Characterization of the Therapeutic Use of AntagonisticSiglec-9 Antibodies and/or Siglec-9 Bispecific Antibodies in a Model ofInflammatory Diseases

The therapeutic utility of antagonistic anti-Siglec-9 and/or Siglec-9bispecific antibodies is tested in a model of inflammatory diseases. Forexample rheumatoid arthritis or in an established model of anotherinflammatory disease (Mizoguchi (2012) Prog Mol Biol Transl Sci.,105:263-320; and Asquith et al., (2009) Eur J Immunol. 39:2040-4). Suchexperiment can be also conducted in mice that express the human Siglec-9gene from a bacterial artificial chromosome or from a cDNA driven by amyeloid promoter or in mice that were transduced with lenti or AAV viruscontaining hSiglec-9 cDNA.

Example 24: Screening for Anti-Siglec-9 Antibodies and/or Siglec-9Bispecific Antibodies that Induce or Inhibit Phosphorylation of Siglec-9and Downstream Signaling Molecules

Cells (J774, RAW 264.7, BMM cells, human primary monocytes, macrophages,neutrophils, NK cells, dendritic cells, T cells, microglia, orosteoclasts) are removed from tissue culture dishes with PBS-EDTA,washed with PBS, and counted. Cells are incubated with an anti-Siglec-9antibodies and/or Siglec-9 bispecific antibody or with anisotype-matched control antibody at 1 μg/10⁶ cells for 20 min on ice orunder other conditions. Cells are lysed in ice-coldradioimmunoprecipitation assay (RIPA) buffer for 20 min followed bycentrifugation at 16,000 g for 10 min at 4° C. to remove insolublematerials. The resulting supernatant is subjected to immunoprecipitationreactions with the indicated antibodies (DAP12, ERK, or AKT) and proteinA- or protein G-agarose (Sigma). The beads are extensively washed withRIPA buffer and the proteins are separated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). The proteins are then transferred tonitrocellulose membranes by Western blotting, incubated with theappropriate antibodies (antibodies that specifically recognizephosphorylated tyrosine or phosphorylated form of DAP12, ERK, Syk, LCK,FYN, C-Cbl, VAV, or AKT) and visualized with the enhancedchemiluminescence (ECL) system (Pierce), as described (e.g., Peng etal., (2010) Sci Signal., 3(122): ra38).

Example 25: Screening for Anti-Siglec-9 and/or Siglec-9 BispecificAntibodies that Induce or Inhibit Calcium Flux

BMM cells are washed twice with HEPES-containing buffer [20 mM HEPES (pH7.3), 120 mM NaCl, 1 mM CaCl, 1 mM MgCl, 5 mM KCl, glucose (1 mg/ml),bovine serum albumin (1 mg/ml)] followed by incubation in 0.05% PluronicF-127 (Invitrogen) and 1 μM Indo-1 AM (Invitrogen) for 20 min at 37° C.Cells are washed twice with HEPES buffer and are then stimulated with ananti-Siglec-9 antibodies and/or Siglec-9 bispecific antibody (16 μg/ml)or with a control antibody (16 μg/ml) and monitored by spectrophotometer(PTL Photon Technology International). The Indo-1 fluorescence emissionis converted to calcium (Ca²⁺) according to manufacturer's instructions(e.g., Peng et al., (2010) Sci Signal., 3(122): ra38).

Example 26: Siglec-9 Increases the Survival of Macrophages, Neutrophils,NK Cells, and Dendritic Cells

To evaluate the role of Siglec-9 in cell survival, human or mousemacrophages, neutrophils, NK cells, microglia, T cells, and dendriticcells are cultured in the presence of inflammatory mediators, and cellsurvival is measured.

Murine bone marrow precursor cells from Siglec-9-expressing or WT miceare obtained by flushing tibial and femoral marrow cells with cold PBS.After one wash with PBS, erythrocytes are lysed using ACK Lysing Buffer(Lonza), washed twice with PBS and suspended at 0.5×10⁶ cells/ml incomplete RPMI media (10% FCS, Pen/Strep, Gln, neAA) with the indicatedamounts of 50 ng/ml M-CSF to produce macrophages, neutrophils, or NKcells, or 10 ng/ml GM-CSF to produce dendritic cells. For M2-typemacrophages, 10 ng/ml IL-4 is added to the cultured cells. For M1-typemacrophages, 50 ng/ml IFN is added. In some experiments LPS or zymosanis added to the cell culture at day 5 at a concentration range of 1μg/ml-0.01 ng/ml. Recombinant cytokines are purchased from Peprotech.

To analyze viability of bone marrow-derived macrophages, neutrophils, orNK cells, cells are prepared as above and cultured in MCSF. Cells areeither plated at 10⁵/200 ul in a 96-well plate (for viability analysisusing a luciferase based-assay) or at 0.5×10⁶/1 ml in a 6-well plate(for Tripan Blue exclusion cell count) in non-tissue culture treatedplates. Media containing fresh M-CSF is added at day 3. At the indicatedtime points cells are gently detached from the plates with 3 mM EDTA andcounted using a Burker chamber. For FACS analysis of live cells,macrophages are cultured either in 50 ng/ml MCSF for 6 days (+MCSF) orin 50 ng/ml MCSF for 4 days before MCSF is removed for an additional 36hrs (−MCSF). Cells are stained using CD11b antibody and DAPI. Forluciferase viability assays, cell viability is measured at day 5 ofculture in graded concentrations of growth factors GMCSF (dendriticcells), MCSF (M1 macrophages), or MCSF+IL-4 (M2 macrophages). Cells aredirectly incubated with ToxGlo reagent (Promega) and luciferase activity(luminescence) is determined. For FACS analysis of viable macrophagescultured in the presence of inflammatory mediators IFN, LPS, or zymosan,cells are collected at day 5 and stained using CD11b antibody and DAPI.

Example 27: Siglec-9 Increases the Expression of Inflammatory CellSurface Markers on Macrophages, Neutrophils, or NK Cells

In order to determine the role of Siglec-9 in inflammatory markerexpression, macrophages, neutrophils, and NK cells are cultured withvarious inflammatory mediators, and the expression of surface markers(e.g., CD86 and CD206) is measured in the presence or absence ofSiglec-9 antibodies.

Macrophages, neutrophils, and NK cells are plated and allowed to adherefor 4 h at 37° C., and TLR agonists LPS (Salmonella abortus equi) andzymosan (Saccharomyces cerevisiae) are added at concentrations rangingfrom 0.01-100 ng/ml (LPS) or 0.01-10 μg/ml (zymosan). Alternatively,macrophages, neutrophils, and NK cells are cultured in the presence ofthe cytokines IL-4 (10 ng/ml) or IFN (0.5-50 ng/ml). FACS analysis ofCD86 and CD206 is performed on a BD FACS Canto 48 hours later. Dataanalysis is performed with FlowJo (TreeStar) software version 10.0.7.

Example 28: Analysis of the Anti-Cancer Effect of Siglec-9 Antibodiesand/or Bispecific Antibodies

Groups of 10 C57B16/NTac mice at 8 weeks (+/−2 weeks) of age, eitherregular mice or mice that express the human Siglec-9 gene from abacterial artificial chromosome or from a myeloid promoter, arechallenged subcutaneously with tumor cells (e.g. 1×10⁵ to 1×10⁶ MC38,Lewis Lung, or B16 cells) suspended in 100 ul PBS. Animals areanesthetized with isoflurane prior to implant. Starting at day 2, groupsof mice are injected i.p. every 3 days for 4 doses with 200 ug of eachof antagonistic anti-Siglec-9 antibodies. Tumor growth is monitored witha caliper biweekly to measure tumor growth starting at day 4. Theendpoint of the experiment is a tumor volume of 2000 nm³ or 60 days.Tumor growth and % survival are the outcome measures. Reduced tumor takeand growth rate, reduced number of tumor infiltrating immune suppressormacrophages, neutrophils, and/or NK cells, and increased effector T cellinflux into the tumor indicate the anti-cancer effects of blockinganti-Siglec-9 antibodies.

Immunodeficient mice or immunodeficient transgenic mice that expresshuman IL-3, human GM-CSF, human IL-6, human IL-2, and were seeded withhuman immune cells from human placenta, fetal liver, peripheral blood oranother source can also be used for such studies (Ito M et al., (2008)Curr Top Microbiol Immunol.; 324:53-76; Ito R., et al., (2012) Cellular& Molecular Immunology 9, 208-214; Brehm et al., (2010) Curr OpinEndocrinol Diabetes Obes. 17(2): 120-125; Zhou et al., (2013) CancerLetters. 344, 13-19). Such mice can be used in conjunction with eithercell line tumors or patient-derived human tumor xenografts (Siolas etal., (2013) Cancer Res.; 73(17): 5315-5319).

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 29: Analysis of Additive Anti-Tumor Effect of CombinationTherapy that Combines Siglec-9 Antibodies and/or Bispecific Antibodieswith Antibodies Against Inhibitory Checkpoint Proteins or InhibitoryCytokines/Chemokines and their Receptors

Groups of 15 C57B16/NTac mice at 8 weeks (+/−2 weeks) of age arechallenged subcutaneously with tumor cells as described in Example 28.Animals are anesthetized with isoflurane prior to implant. Starting atday 2, mice are injected i.p. every 3 days for 4 doses with 200 uganti-Siglec-9 antibodies alone or in combination with antibodies againstcheckpoint proteins (e.g. anti-PDL1 mAb clone 10F.9G2 and/or anti-CTLA4mAb clone UC10-4F10-11) at day 3, 6, and 9. Treatment groups includeanti-Siglec-9; anti-CTLA4; anti-PDL1; anti-Siglec-9+anti-CTLA4;anti-Siglec-9+anti-PDL1; and isotype control. Tumor growth is monitoredwith a caliper biweekly to measure tumor growth starting at day 4. Theendpoint of the experiment is a tumor volume of 2000 nm³ or 60 days.Tumor growth and % survival are the outcome measures. A decrease intumor growth and an increase in % survival with combination therapyindicate that anti-Siglec-9 antibodies have additive or synergistictherapeutic effects with anti-checkpoint antibodies. Antagonisticantibodies against checkpoint molecules include antibodies against PDL1,PDL2, PD1, CTLA4, B7-H3, B7-H4, HVEM, LIGHT, BTLA, KIR, GAL9, TIM1,TIM3, TIM4, CD39, CD73, TIGIT, VISTA, A2AR, LAG-3, and PhosphatidylSerine. Antagonist antibodies against inhibitory cytokines includeantibodies against CCL2, CSF-1, and IL-2. Immuno-deficient mice orimmuno-deficient transgenic mice that express human IL-3, human GM CSF,human IL-6, human IL-2, and were seeded with human immune cells fromhuman placenta, fatal liver, peripheral blood or another source can alsobe used for such studies (Ito M et al., (2008) Curr Top MicrobiolImmunol.; 324:53-76; Ito R., et al., (2012) Cellular & MolecularImmunology 9, 208-214; Brehm et al., (2010) Curr Opin EndocrinolDiabetes Obes. 17(2): 120-125; Zhou et al., (2013) Cancer Letters. 344,13-19). Such mice can be used in conjunction with either cell linetumors or Patient derived human tumors xenografts (Siolas et al., (2013)Cancer Res.; 73(17): 5315-5319).

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 30: Analysis of Additive Anti-Tumor Effect of CombinationTherapy that Combines Siglec-9 Antibodies and/or Bispecific Antibodieswith Antibodies that Activate Stimulatory Checkpoint Proteins

Groups of 15 C57B16/NTac mice at 8 weeks (+/−2 weeks) of age arechallenged subcutaneously with tumor cells as described in Example 28.Animals are anesthetized with isoflurane prior to implant. Starting atday 2, mice are injected i.p. every 3 days for 4 doses with 200 uganti-Siglec-9 antibodies alone or in combination with agonisticantibodies that activate stimulatory checkpoint proteins (e.g. OX40 orICOS mAb) at day 3, 6, and 9. Tumor growth is monitored with a caliperbiweekly to measure tumor growth starting at day 4. The endpoint of theexperiment is a tumor volume of 2000 mm³ or 60 days. Tumor growth and %survival are the outcome measures. A decrease in tumor growth and anincrease in % survival with combination therapy indicate thatanti-Siglec-9 antibodies have additive or synergistic therapeuticeffects with stimulatory checkpoint antibodies. Stimulatory checkpointantibodies include agonistic/stimulatory antibodies against CD28, ICOS,CD137, CD27, CD40, and GITR.

Immuno-deficient mice or immuno-deficient transgenic mice that expresshuman IL-3, human GM CSF, human IL-6, human 112, and were seeded withhuman immune cells from human placenta, fatal liver, peripheral blood oranother source can also be used for such studies (Ito M et al., (2008)Curr Top Microbiol Immunol.; 324:53-76; Ito R., et al., (2012) Cellular& Molecular Immunology 9, 208-214; Brehm et al., (2010) Curr OpinEndocrinol Diabetes Obes. 17(2): 120-125; Zhou et al., (2013) CancerLetters. 344, 13-19). Such mice can be used in conjunction with eithercell line tumors or Patient derived human tumors xenografts (Siolas etal., (2013) Cancer Res.; 73(17): 5315-5319).

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced withlentivirus or AAV virus containing human Siglec-9 cDNA.

Example 31: Analysis of Additive Anti-Tumor Effect of CombinationTherapy that Combines Siglec-9 Antibodies and/or Bispecific Antibodieswith Stimulatory Cytokines

Groups of 15 C57B16/NTac mice at 8 weeks (+/−2 weeks) of age arechallenged subcutaneously with tumor cells as described in Example 28.Animals are anesthetized with isoflurane prior to implant. Starting atday 2, mice are injected i.p. every 3 days for 4 doses with 200 uganti-Siglec-9 antibodies alone or in combination with stimulatorycytokines (e.g. IL-12, IFN-a). Tumor growth is monitored with a caliperbiweekly to measure tumor growth starting at day 4. The endpoint of theexperiment is a tumor volume of 2000 mm³ or 60 days. Tumor growth and %survival are the outcome measures. A decrease in tumor growth and anincrease in % survival with combination therapy indicate thatanti-Siglec-9 antibodies have additive or synergistic therapeuticeffects with immune-stimulatory cytokines. Stimulatory cytokines includeIFN-α/b, IL-2, IL-12, IL-18, GM-CSF, and G-CSF.

Immuno-deficient mice or immuno-deficient transgenic mice that expresshuman IL-3, human GM CSF, human IL-6, human 112, and were seeded withhuman immune cells from human placenta, fatal liver, peripheral blood oranother source can also be used for such studies (Ito M et al., (2008)Curr Top Microbiol Immunol.; 324:53-76; Ito R., et al., (2012) Cellular& Molecular Immunology 9, 208-214; Brehm et al., (2010) Curr OpinEndocrinol Diabetes Obes. 17(2): 120-125; Zhou et al., (2013) CancerLetters. 344, 13-19). Such mice can be used in conjunction with eithercell line tumors or Patient derived human tumors xenografts (Siolas etal., (2013) Cancer Res.; 73(17): 5315-5319).

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 32: Analysis of Ability of Siglec-9 Antibody and/or BispecificAntibody Fabs to Stimulate Viability of Innate Immune Cells

The agonistic functionality of plate bound, cross-linked anti-Siglec-9antibody Fab fragments is evaluated in innate immune cells (e.g.,macrophages, neutrophils, and NK cells).

Macrophages, neutrophils, and NK cells are cultured in the presence ofM-CSF and plate bound Siglec-9 antibody Fabs, and cell viability ismeasured.

Macrophages, neutrophils, NK cells, and DC derived from human monocytes,as well as T cells and human microglia derived from human monocytes areplated on non-tissue-culture-treated 96-well plates, pre-coated witheither 12.5 nM or 100 nM of cross-linked Siglec-9 Fabs. Cells arecultured for 48 hours in the presence of 10 ng/ml M-CSF. Analysis ofviability is performed using CellTiter-Glo® kit (Promega). Plates areread with a BioTek Synergy Microplate Reader using GEN5 2.04 software.

Example 33: Analysis of the Ability of Siglec-9 Antibodies and/orBispecific Antibodies to Modulate NFAT-Dependent Genes

The ability of antagonistic anti-Siglec-9 antibodies activateNFAT-dependent genes is evaluate using a luciferase reporter gene underthe control of an NFAT (nuclear factor of activated T cells) promoter.

A cell line derived from mouse T lymphocytes BW5147.G.1.4 (ATCC® TIB48™)that express the ITAM motif containing co-receptor DAP12 and its ligandbinding partner TREM2 is infected with human Siglec-9, and with CignalLenti NFAT-Luciferase virus (Qiagen). Luciferase signaling is activatedby plate bound anti-TREM2 antibodies. Full-length and Fab fragmentanti-Siglec-9 antibodies are either co-plated with the TREM2 antibodiesor applied in solution. For plate binding, antibodies are applied at 10μg/ml in DPBS on tissue-culture treated clear bottom white 96 wellplates (100 ul/well), overnight at 4° C. Wells are rinsed three timeswith DPBS and subsequently plated at 100,000 cells/well in media with 1%serum. As a positive control for signaling, PMA (0.05 ug/ml) andionomycin (0.25 uM) are added together. Cells are incubated for 6 hoursand luciferase activity is measured by adding ONE-Glo™ reagent (Promega)to each well and incubating 3 min at RT on a plate shaker. Luciferasesignal is measured using a BioTek plate reader.

Example 34: Analysis of Anti-Stroke Effect of Siglec-9 Antibodies and/orBispecific Antibodies

Transient occlusion of the middle cerebral artery (MCAO)—a model thatclosely resembles human stroke is used to induce cerebral infarction inmice. Monofilament (70SPRe, Doccol Corp, USA) is introduced into theinternal carotid artery through an incision of the right common carotidartery. The middle cerebral artery is occluded for 30 minutes with arange of reperfusion times (6 h, 12 h, 24 h, 2 d, 7 d and 28 d). Theeffect of surgery is controlled using sham animals at 12 h and at 7 d.Sham animals undergo the same surgical procedure without occlusion ofthe middle cerebral artery. MCAO animals treated with antagonisticanti-Siglec-9 antibodies or control antibodies are tested for infarctvolumetry, acute inflammatory response (12 h reperfusion), transcriptionof pro-inflammatory cytokines TNFa, IL-1a, and IL-1b, microglialactivity (CD68, Iba1), transcription of chemokines CCL2 (MCP1), CCL3(MIP1a and the chemokine receptor CX3CR1 and invasion of CD3-positive Tcells (Sieber et al. (2013) PLoS ONE 8(1): e52982.doi:10.1371/journal.pone.0052982.). Such experiment can be conducted inregular mice or alternatively in mice that express the human Siglec-9gene from a bacterial artificial chromosome or from a cDNA driven by amyeloid promoter or in mice that were transduced with lenti or AAV viruscontaining hSiglec-9 cDNA.

Example 35: Analysis of Anti-Alzheimer's Disease Effect of Anti-Siglec-9Antibodies and/or Bispecific Antibodies

To evaluate the ability of antagonistic anti-Siglec-9 antibodies todelay, prevent, or reverse the development of Alzheimer's disease (AD),5×FAD mice are used. 5×FAD mice overexpress mutant human APP (695) withthe Swedish (K670N, M671L), Florida (I716V), and London (V717I) familialAlzheimer's disease (FAD) mutations, along with human PS1 harboring twoFAD mutations, M146L and L286V. Both transgenes are regulated by themouse Thy1 promoter to drive over expression on the brain andrecapitulate major features of AD. Mice treated with the agonisticanti-Siglec-9 antibodies or with control antibodies are tested for Abeta plaque load with immunohistochemistry and by ELISA of tissueextracts. They are further tested for the number of microglia in thebrain, and for reduction in cognitive deficit using Morris Water maze, aspatial learning and memory task, Radial Arm Water Maze, a spatiallearning and memory task, Y Maze (quantifies spontaneous alternation asa measure of spatial cognition), novelty preference in an open field,operant learning to assess learning and memory, and fear conditioning(mousebiology.org website; Wang et al., (2015) Cell. pii:S0092-8674(15)00127-0). Such experiment can be also conducted in micethat express the human Siglec-9 gene from a bacterial artificialchromosome or from a cDNA driven by a myeloid promoter or in mice thatwere transduced with lenti or AAV virus containing hSiglec-9 cDNA.

Example 36: Analysis of the Protective Effect of Siglec-9 Antibodiesand/or Bispecific Antibodies in Respiratory Tract Infections

To evaluate the ability of antagonist Siglec-9 antibodies to delay,prevent, or treat bacterial respiratory tract infections, a preclinicalmouse model involving challenge of C57B16 mice with Streptococcuspneumoniae is used. This model involves intranasal (i.n.) administrationof 105 CFU S. pneumoniae serotype 3 (ATCC 6303) as described (see, e.g.,Sharif O et al, 2014 PLoS Pathog. 2014 June; 10(6): e1004167; andSchabbauer G et al, 2010 J Immunol 185: 468-476). In this model ˜90% WTC57B16 mice succumb to infection within 6 days post infection.

Ten to fifteen mice/group are challenged with S. pneumoniae andconcomitantly are treated with antagonist anti-Siglec-9 antibodies everyother day starting from day 0. The first dose of anti-Siglec-9antibodies is administered 3 hours prior to challenge with S. pneumonia.Mice are monitored daily for 15 days to check for death events. % ofmice surviving bacteria challenge is determined.

In separate experiments, count of bacterial load and cytokine expressionin the blood and in the lungs is also determined. 24 or 48 hours afterinfection blood is collected in EDTA-containing tubes and plated on agarplates to enumerate bacterial CFU in the plasma. Plasma is stored at−20° C. for cytokine analysis by ELISA. Lungs are harvested, homogenizedand plated on agar plates to enumerate bacterial CFU, or incubated for30 min in lysis buffer and supernatants analyzed for cytokinemeasurements.

In separate experiments, lungs are collected 40 hours post bacterialinfection, fixed in 10% formalin, and embedded in paraffin for H&Epathology analysis.

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 37: Analysis of the Protective Effect of Siglec-9 Antibodiesand/or Bispecific Antibodies in Sepsis

To evaluate the ability of antagonist Siglec-9 antibodies to delay,prevent, or treat sepsis, a preclinical mouse model involving systemicchallenge of C57B16 mice with LPS is used. This model involvesintraperitoneal (i.p.) administration of 37 mg/ml LPS as described (see,e.g., Gawish R et al, 2014 FASEB J). In this model >95% WT C57B16 micesuccumb infection within 40 hours post LPS injection.

Cohorts of mice are challenged with LPS and concomitantly are treatedwith antagonist anti-Siglec-9 antibodies every day starting from day 0.The first dose of anti-Siglec-9 antibodies is administered 3 hours priorto challenge with LPS. Mice are monitored every ˜4 hours during daytime,to check for death events. Percentage of mice surviving LPS challenge isdetermined.

In separate experiments, peritoneal lavage fluid (PLF) is collected.Supernatants are stored at −20° C. for cytokine analysis by ELISA;pelleted cells are counted to quantify inflammatory cells recruited inthe peritoneal cavity. Similar studies can be conducted to test theefficacy of Siglec-9 antibodies in other models of infection (see, e.g.,Sun et al., (2013) Invest Ophthalmol Vis Sci. 17; 54(5):3451-62).

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 38: Analysis of the Protective Effect of Siglec-9 Antibodiesand/or Bispecific Antibodies in Acute and Chronic Colitis

To evaluate the ability of antagonist anti-Siglec-9 antibodies to delay,prevent, or treat colitis, preclinical mouse models of acute or chroniccolitis are used. For DSS-induced colitis, mice receive 3% DSS indrinking water ad libitum for 8 days. For TNBS-induced colitis, mice areanesthetized and treated with an intra-rectal injection of 3 mg TNBS in20% ethanol (vol/vol) or vehicle alone as a control. For the chroniccolitis model, all mice are treated with 3 cycles of 2% DSS for 5 days,followed by a 10-day recovery period. For all models, weight loss, stoolconsistency, and presence of fecal occult blood are monitored daily andused to calculate the disease activity index, as described (see, e.g.,Correale C, 2013, Gastroenterology, February 2013, pp. 346-356.e3).

Cohorts of mice are treated with antagonist anti-Siglec-9 antibodiesevery day starting from day 0 and subjected to DSS or TNBSadministration. Mice are monitored every day, to check for weight loss,stool consistency, and presence of fecal occult blood were monitoreddaily and used to calculate the disease activity index, as described(see, e.g., S. Vetrano, Gastroenterology, 135 (2008), pp. 173-184).

In separate experiments, endoscopic and histological images of mucosaldamage are collected to evaluate inflammatory cell infiltration andmucosal damage. Similar studies can be conducted to test the benefit ofSiglec-9 antibodies in other models of autoimmunity including Crohn'sdisease, inflammatory bowel disease, and ulcerative colitis (see, e.g.,Low et al., (2013) Drug Des Devel Ther.; 7: 1341-1357; and Sollid etal., (2008) PLoS Med 5(9): e198).

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 39: Analysis of the Protective Effect of Siglec-9 Antibodiesand/or Bispecific Antibodies in Wound Healing

To evaluate the ability of agonistic anti-Siglec-9 antibodies toincrease colonic wound repair following injury, a mouse model of biopsyinjury in the colon is used. In this model, the endoscope with outeroperating sheath is inserted to the mid-descending colon and the mucosais surveyed to the ano-rectal junction. Then, a single full thicknessarea of the entire mucosa and submucosa is removed with flexible biopsyforceps with a diameter of 3 French, avoiding penetration of themuscularis propria. Each mouse is biopsy injured at 3-5 sites along thedorsal side of the colon (see, e.g., Seno H, 2008, Proc Natl Acad Sci USA. 2009 Jan. 6; 106(1): 256-261).

Cohorts of mice are treated with agonist anti-Siglec-9 antibodies 2 or 3days after biopsy injury. Mice are monitored every day for 15 days, tocheck for weight loss and wound healing by measuring the surface area oflesions.

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 40: Analysis of the Protective Effect of Siglec-9 Antibodiesand/or Bispecific Antibodies in Retinal Degeneration

AMD is a degenerative disease of the outer retina. It is thought thatinflammation, particularly inflammatory cytokines and macrophages,neutrophils, and/or NK cells contribute to AMD disease progression.

The presence of macrophages, neutrophils, and NK cells in the proximityof AMD lesions is documented, in the drusen, Bruch's membrane, choroidand retina. Macrophages, neutrophils, and NK cells release tissue factor(TF) and vascular endothelial growth factor (VEGF), which triggers theexpansion of new blood vessels formation in patients showing choroidalneovasulcarization.

The type of macrophage present in the macular choroid changes with age,displaying elevated levels of M2 macrophages, neutrophils, and NK cellsin older eyes compared to younger eyes. However, advanced AMD maculaehad higher M1 to M2 rations compared to normal autopsied eyes of similarage. (see, e.g., Cao X et al, (2011), Pathol Int 61(9): pp 528-35). Thissuggests a link between classical M1 macrophage activation in the cyc inthe late onset of AMD progression.

Retinal microglia cells are tissue-resident macrophages that are alsonormally present in the inner retina. In the event of damage, microgliacan be activated and act as mediator of inflammation. Activatedmicroglia has been detected in the AMD tissue samples and has beenproposed as one potential contributor of inflammatory processed thatlead to AMD pathogenesis (Gupta et al., (2003) Exp Eye Res.,76(4):463-71.). The ability of Siglec-9 antibodies to prevent, delay, orreverse AMD is tested in one or more of AMD models (see, e.g., Pennesiet al., (2012) Mol Aspects Med.; 33(4): 487-509).

Overall inflammatory macrophages, neutrophils, and NK cells (either M1and/or activated microglia) are documented to correlate with AMD diseaseprogression and therefore represent a therapeutic target for antagonistSiglec-9 antibodies. Similar therapeutic benefit can be achieved inglaucoma and genetic forms or retinal degeneration such as retinitispigmentosa.

The ability of Siglec-9 antibodies to prevent, delay, or reverse retinalganglion cell degeneration in glaucoma is tested in a glaucoma model(see, e.g., El-Danaf et al., (2015). J Neurosci. 11; 35(6):2329-43).Likewise, the therapeutic benefit of REM2 in genetically induced retinaldegeneration and retinitis pigmentosa is tested as described in Chang etal., (2002) Vision Res.; 42(4):517-25, and in “Retinal Degeneration RatModel Resource Availability of P23H and S334ter Mutant RhodopsinTransgenic Rats and RCS Inbred and RCS Congenic Strains of Rats,” MMLaVail, Jun. 30, 2011.

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 41: Analysis of the Protective Effect of Siglec-9 Antibodiesand/or Bispecific Antibodies in Adipogenesis and Diet-Induced Obesity

To test the effect of Siglec-9 antibodies in adipogenesis and obesity, amouse model of high-fat diet (HFD) is used (see, e.g., Park et al.,(2015) Diabetes. 64(1):117-27).

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 42: Analysis of the Protective Effect of Antagonist Siglec-9Antibodies and/or Bispecific Antibodies in Osteoporosis

Bone is a dynamic organ constantly remodeled to support calciumhomeostasis and structural needs. The osteoclast is the cell responsiblefor removing both the organic and inorganic components of bone. Theosteoclast is derived from hematopoietic progenitors in the macrophagelineage and differentiates in response to the tumor necrosis factorfamily cytokine receptor activators of NFκB ligand. Osteoclasts, theonly bone-resorbing cells, are central to the pathogenesis ofosteoporosis and osteoporosis (Novack et al., (2008) Annual Rev Pathol.,3:457-84).

Osteoporosis is a progressive bone disease that is characterized by adecrease in bone mass and density which can lead to an increased risk offracture. It is mostly manifested in the first years followingmenopause, when bone turnover is accelerated, with increased activity ofboth osteoclasts and osteoblasts. Owing to an imbalance in the processesof resorption and synthesis, however, the net effect is bone loss, whichis largely trabecular. Thus, the most prevalent sites of fracture inosteoporosis are the wrist, femoral neck, and vertebral bodies, in whichthe trabecular structure is key to overall bone strength.

Reduced osteoclast function results in osteoporosis, with increased bonemass and elimination of bone marrow space, as observed in animal modelslacking DAP12 ITAM signaling adapter and resulting in a significantdefect in differentiation of osteoclast-like cells (Koga, et al., (2004)Nature 428: 758-763).

Thus, administering an antagonist anti-Siglec-9 antibody of the presentdisclosure can prevent, reduce the risk of, and/or treat osteoporosis.In some embodiments, administering an agonist anti-Siglec-9 antibody mayinduce one or more Siglec-9 activities in an individual havingosteoporosis (e.g., DAP12 phosphorylation, Syk activation, andaccelerated differentiation into osteoclasts) (Peng et al (2010). SciSignal. 2010 18; 3 122; and Humphrey et al., (2006) J Bone Miner Res.,21(2):237-45).

Such experiment can be also conducted in mice that express the humanSiglec-9 gene from a bacterial artificial chromosome or from a cDNAdriven by a myeloid promoter or in mice that were transduced with lentior AAV virus containing hSiglec-9 cDNA.

Example 43: Analysis of the Ability of Siglec-9 Antibodies and/orBispecific Antibodies to Modulate Binding of Siglec-9 to SHP1, SHP2 andOther Signaling Molecules

Human primary monocytes, macrophages, neutrophils, NK cells, dendriticcells, T cells, microglia or osteoclasts are removed from tissue culturedishes with PBS-EDTA, washed with PBS, and counted. Cells are incubatedwith an anti-Siglec-9 and/or Siglec-9 bispecific antibody or with anisotype-matched control antibody at 1 μg/10⁶ cells for 20 min on ice orunder other conditions. Cells are lysed in ice-coldradioimmunoprecipitation assay (RIPA) buffer for 20 min followed bycentrifugation at 16,000 g for 10 min at 4° C. to remove insolublematerials. The resulting supernatant is subjected to immunoprecipitationreactions with the indicated antibodies (SHP1, SHP2, c-Cbl., Vav, Syk,LcK, Fyn, GRb2, PLC-gamma. Toll like receptor, DAMP receptors, patternrecognition receptor) and protein A- or protein G-agarose (Sigma). Thebeads are extensively washed with RIPA buffer and the proteins areseparated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Theproteins are then transferred to nitrocellulose membranes by Westernblotting, incubated with the appropriate Siglec-9 antibodies andvisualized with the enhanced chemiluminescence (ECL) system (Pierce), asdescribed (e.g., Peng et al., (2010) Sci Signal., 3(122): ra38).Alternatively, the cells are incubated with an anti-Siglec-9 and/orSiglec-9 bispecific antibody or with an isotype-matched control antibodyat 1 μg/10⁶ cells for 20 min on ice or under other conditions. Cells arelysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer for 20min followed by centrifugation at 16,000 g for 10 min at 4° C. to removeinsoluble materials. The resulting supernatant is subjected toimmunoprecipitation reactions with a second Siglec-9 antibody. Theproteins are then transferred to nitrocellulose membranes by Westernblotting and incubated with the indicated antibodies (SHP1, SHP2,c-Cbl., Vav, Syk, LcK, Fyn, GRb2, PLC-gamma. Toll like receptors, DAMPreceptors, and pattern recognition receptors).

Example 44: Analysis of the Ability of Siglec-9 Antibodies and/orBispecific Antibodies to Enhance Tumor Cell Killing by NK Cells

Natural killer cells are isolated from human blood using RosetteSepHuman NK Cell Enrichment Cocktail (STEMCELL Technologies). Isolatedcells are cultured in RPMI 1640 (Mediatech) supplemented with 10%Hyclone Fetal Bovine Serum (GE Healthcare), 2% Hepes, 2% GlutaMAX, 2%penicillin-streptomycin, 2% sodium pyruvate, and 2% MEM non-essentialamino acids (Life Technologies). NK cells are plated 1×10⁶ cells/wellwith 25 ng/ml IL-2 (R&D) and 25 ng/nil IL-21 (Peprotech) in 24-wellplates and incubated at 37° C. with 5% CO₂ for 7 days. NK cells arephenotyped at isolation and day 7 with APC anti-human CD56 (Biolegend)PE-Cy7 anti-human CD3 (Biolegend), and V500 mouse anti-human CD16 (BDBiosciences).

Day 7 post culture, NK cells are harvested and resuspended at 1×10⁶cells/ml. NK effector cells are plated at Effector:Target ratios of12:1, 4:1, 2:1, 0.5:1 and at 120,000 cells for effector only controlwells in 96-well U-bottom plates. Cells are centrifuged and resuspendedin 100 ul media (50 ul 100% EtOH and 50 ul media are added to positivecontrol wells to serve as 100% killing). 10 μg/ml Siglec-7antibodies/mIgG1 isotype control are added, mixed, and incubated on icefor 20 mins. 10,000 CFSE-labeled K562 target cells are added to eachwell, excluding effector only control wells. The plate is centrifuged at1500 rpm for 1 min and incubated at 37° C. with 5% CO₂ for 2 hr.Following incubation, the cells are stained with 50 ul NucBlue FixedCell ReadyProbes Reagent (Life Technologies), incubated at RT protectedfrom light for 5 mins and analyzed by flow cytometry on FACS Canto (BDBiosciences) immediately.

Example 45: Analysis of Siglec-9 Antibody Binning

Monocytes were isolated from human blood samples using RosetteSep™ HumanMonocyte Enrichment Cocktail (STEMCELL Technologies, Vancouver, Canada).Isolated cells were cultured in RPMI 1640 (Mediatech) supplemented with10% HyClone™ FBS (GE Healthcare Life Sciences), 20 mM HEPES, 4 mMGlutaMAX, 200 U/ml Penicillin-Streptomycin, 2 mM Sodium Pyruvate, and 2%MEM NEAA (Life Technologies). Cells were at plated 1×10⁶ cells/ml with8% human serum (Sigma-Adrich) and 50 ng/ml recombinant human M-CSF(PeproTech) in T150 tissue culture flasks and incubated at 37° C. with5% CO₂ for 5 days.

At day 5, adherent monocyte-derived macrophages were harvested andplated at 100,000 cells/well in a 96-well U-bottom non-TC treated plate.5 μg/ml purified Siglec-9 antibodies were incubated with cells in thepresence of human Fc receptor binding inhibitor (eBioscience) in PBSwith 1% BSA and 2 mM EDTA. Samples were incubated on ice for 30 min andwashed 2× in buffer. Direct fluorophore conjugated Siglec-9 antibodieswere added at 5 μg/ml and incubated on ice for 30 min followed by 2washes in buffer. Ability of the conjugated Siglec-9 antibody clones tobind cell surface receptor in the presence of bound Siglec-9 antibodies,isotypes, or untreated were determined by flow cytometry on an iQueScreener (IntelliCyt) and analyzed with FlowJo software (TreeStar) todetermine competition of the clones. The conjugated anti-Siglec-9antibodies used were: E10286 (BD Biosciences), K8 (BioLegend), and191240 (R&D Systems). Bound anti-Siglec-9 antibodies used were: 2D4,2D5, 5B1, 6B2, 6D8, 7H12, 5C6, 12B12, and 17C2.

The results are depicted in Table 12.

TABLE 12 Siglec-9 antibody binning Bin 1 Bin 2 Bin 3 Bin 4 Bin 5Competes Competes Competes Competes Does not with with K8 with withcompete with E10286 191240 E10286, K8, any tested and 191240 antibody Ab2D4 5C6 6B2 5B1 2D5 12B12 6D8 7H12 17C2 5C6 17C2

The results indicate that Siglec-9 antibodies 5C6, 12B12, and 17C2 arein a distinct bin from antibodies 2D4, 2D5, 5B1, and 7H12. The resultsare consistent with those in Example 1.

Example 46: In Vivo Effects of Anti-Siglec-9 and Anti-PD1 AntibodyCombination Treatment

A CTG-0202 patient-derived melanoma tumor was first passaged inpre-study animals prior to implantation to humanized mice. When tumorsreached 1-1.5 cm³ in stock animals, they were harvested forre-implantation into pre-study animals. Pre-study animals were implantedunilaterally on the left flank with tumor fragments. The CTG-0202melanoma tumors were used at passage number 6.

Immunocompromised female mice (Taconic NOG) were humanized with fetalliver-derived CD34+ hematopoietic cells. Mice were housed on irradiatedpapertwist-enriched 1/8″ corncob bedding (Sheperd) in individual HEPAventilated cages (Innocage® IVC, Innovive USA) on a 12-hour light-darkcycle at 68-74° F. (20-23° C.) and 30-70% humidity. Mice were fed waterad libitum (reverse osmosis, 2 ppm Cl₂) and an irradiated test rodentdiet (Teklad 2919) consisting of 19% protein, 9% fat, and 4% fiber. 48humanized mice were implanted, and pre-study tumor volumes were recordedfor each experiment beginning seven to ten days after implantation. Whentumors reached approximately 80-200 mm³ in volume, mice were matched bytumor volume into treatment or control groups to be used for dosing, anddosing was initiated on Day 0. Test groups were administered anti-PD-1antibody Keytruda® (pembrolizumab) in combination with a controlmonoclonal antibody (Group 1), or in combination with anti-Siglec-9antibody 2D4 (Group 2). Table 13 and FIG. 15A depict the dosingschedule, dosing amounts, and route of administration.

TABLE 13 Antibody dosing schedule and route of administration for the invivo cancer efficacy study of the Siglec-9 antibody 2D4 Antibody DoseVolume Total # Group n Treatment (mg/kg) ROA Schedule of Doses 1 8Keytruda 5/2.5 IP q5dx6 6 CTR mAb 40 IP q5dx6 6 2 8 Keytruda 5/2.5 IPq5dx6 6 2D4 40 IP q5dx6 6 In Table 13, “n” refers to the number of micein each treatment group; “CTR mAb” refers to an isotype controlantibody; “2D4” refers to anti-Siglec-9 antibody 2D4; “ROA” refers toroute of administration; “IP” refers to intraperitoneal; “q5dx6” refersto a dosing schedule of administration every five days for a total ofsix doses.

Beginning at Day 0, mice were observed daily and weighed twice weeklyusing a digital scale. Tumor dimensions were measured twice weekly bydigital caliper and data including individual and mean estimated tumorvolumes (Mean TV±SEM) was recorded for each group. Tumor volume wascalculated using the formula (1): TV=width2×length×0.52. The study wasconcluded when the mean tumor volume of the control group reached 1500mm³ at day 28.

At study termination harvested tumors were shipped overnight in media onice packs and processed the following day. Tumor samples were treatedwith collagenase for 30 min at 37° C. Samples were dissociated through acell strainer and resuspended in 2% FBS in PBS. Red blood cells in wholeblood samples were lysed using ACK lysing buffer and cells were thenwashed in 2% FBS in PBS twice. Cells were counted using a hemocytometerand one million cells were stained with fluorochrome-conjugatedantibodies for 30 minutes on ice, then washed with 2% FBS in PBS. Cellswere fixed with 4% paraformaldehyde in PBS. All the stained cells wereanalyzed on a FACS Canto (BD Biosciences) and the data analyzed withFlowJo software (TreeStar). To identify tumor-infiltrating immune cells,hCD45, hCD3, hCD4, hCD8, and hCD14 antibodies were used to gate onpopulations according to standard procedures.

As shown in FIGS. 15B and 15C, the combination treatment with theSiglec-9 antibody 2D4 and the anti-PD-1 antibody Keytruda®(pembrolizumab) reduced cell surface levels of Siglec-9 in peripheralblood hCD45⁺CD14⁺ myeloid cells in a mouse tumor model that wasengrafted with human immune stem cells and the patient-derived xenograftCTG-0202 melanoma tumors. 2D4 treatment did not impact the levels ofcell surface CD33. A similar reduction in cell-surface Siglec-9 levelswas also observed on intratumoral hCD45⁺CD14⁺ myeloid cells.

As shown in FIG. 15D the combination treatment with the Siglec-9antibody 2D4 and the anti-PD-1 antibody Keytruda® (pembrolizumab)reduced the percentage of peripheral blood hCD45⁺CD14⁺ myeloid cells ina mouse tumor model that was engrafted with human immune stem cells andthe patient-derived xenograft CTG-0202 melanoma tumors.

As shown in FIG. 15E the combination treatment with the Siglec-9antibody 2D4 and the anti-PD-1 antibody Keytruda® (pembrolizumab)increased the percentage of peripheral blood hCD45⁺ CD3⁺ T cells in amouse tumor model that was engrafted with human immune stem cells andthe patient-derived xenograft CTG-0202 melanoma tumors.

As shown in FIG. 15F, the combination treatment with the Siglec-9antibody 2D4 and the anti-PD-1 antibody Keytruda® (pembrolizumab)reduced the number of tumor infiltrating hCD45⁺CD14⁺ myeloid cells in amouse tumor model that was engrafted with human immune stem cells andthe patient-derived xenograft CTG-0202 melanoma tumors.

As shown in FIG. 15G, the combination treatment with the Siglec-9antibody 2D4 and the anti-PD-1 antibody Keytruda® (pembrolizumab)increased the number of tumor infiltrating hCD45⁺CD3⁺ T cells in a mousetumor model that was engrafted with human immune stem cells and thepatient-derived xenograft CTG-0202 melanoma tumors.

As shown in FIG. 15H-15J, the combination treatment with the Siglec-9antibody 2D4 and the anti-PD-1 antibody Keytruda® (pembrolizumab)inhibited tumor growth in vivo in a patient-derived melanoma tumor modelthat was implanted in mice engrafted with human immune stem cells fromtwo different human donors (Donor #165547112, and 17509112), while 2D4treatment did not impact the growth of tumors in mice engrafted withimmune cells from a third donor (Donor #984480112).

Importantly for the results described herein, both the tumor and theimmune system responding to the tumor were human. The immune system ofthe mouse was humanized using a method whereby the mouse immune systemwas genetically ablated and myeloablated with irradiation, then replacedwith human donor CD34⁺ hematopoietic stem and progenitor cells thatrepopulated and developed myeloid and lymphoid immune cells. The tumorwas a patient-derived melanoma. The human-specific Siglec-9 antibody 2D4displayed target specific engagement and downregulation of humanSiglec-9. Receptor downregulation was significant on peripheral andtumor CD14⁺ myeloid cells. Therefore any effects on tumor growth,increased tumor infiltration of CD3⁺ T-cells, and reduced CD14+ cellswere the result of 2D4 antibody-mediated downregulation of Siglec-9 onhuman myeloid immune cells.

The data presented above showed that the Siglec-9 antibody 2D4 was apotent and significant receptor-downregulating antibody on peripheraland tumor infiltrating myeloid cells. Analysis of both circulating andtumor infiltrating immune cell populations demonstrated an increase ofCD3⁺ T-cells. Additionally, myeloid cell populations were also affectedby Siglec-9 antibody treatment. A decrease in CD14⁺ myeloid cells wasobserved in tumors, as well as in peripheral blood cell populations.This data suggested that downregulation of Siglec-9 functionally alteredtumor infiltrating immune cell populations. Taken together, thesestudies supported the pre-clinical efficacy of anti-Siglec-9 antibodiesas a therapeutic for treating human cancer.

What is claimed is:
 1. An isolated anti-Siglec-9 antibody, wherein theanti-Siglec-9 antibody comprises a light chain variable domain and aheavy chain variable domain, wherein the light chain variable domaincomprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 6or an amino acid sequence with at least 90% homology to SEQ ID NO: 6, anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 10 or an aminoacid sequence with at least 90% homology to SEQ ID NO: 10, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 14 or an amino acidsequence with at least 90% homology to SEQ ID NO: 14, and the heavychain variable domain comprises an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 19 or an amino acid sequence with at least 90%homology to SEQ ID NO: 19, an HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 22 or an amino acid sequence with at least 90% homology toSEQ ID NO: 22, and an HVR-H3 comprising the amino acid sequence of SEQID NO: 26 or an amino acid sequence with at least 90% homology to SEQ IDNO:
 26. 2. The anti-Siglec-9 antibody of claim 1, wherein theanti-Siglec-9 antibody comprises an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 6, an HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 10, an HVR-L3 comprising the amino acid sequence of SEQ IDNO: 14, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19,an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and anHVR-H3 comprising the amino acid sequence of SEQ ID NO:
 26. 3. Theanti-Siglec-9 antibody of claim 2, wherein the antibody is of the IgGclass the IgM class, or the IgA class.
 4. The anti-Siglec-9 antibody ofclaim 3, wherein the anti-Siglec-9 antibody has an IgG1, IgG2, IgG3, orIgG4 isotype.
 5. The anti-Siglec-9 antibody of claim 4, wherein: (a) theanti-Siglec-9 antibody has a human or mouse IgG1 isotype and comprisesone or more amino acid substitutions in the Fc region at a residueposition selected from the group consisting of: N297A, D265A, D270A,L234A, L235A, G237A, P238D, L328E, E233D, G237D, H268D, P271G, A330R,C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L,M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A,T394D, and any combination thereof, wherein the numbering of theresidues is according to EU numbering, or comprises an amino aciddeletion in the Fc region at a position corresponding to glycine 236;(b) the anti-Siglec-9 antibody has an IgG1 isotype and comprises an IgG2isotype heavy chain constant domain 1(CH1) and hinge region, optionallywherein the IgG2 isotype CH1 and hinge region comprises the amino acidsequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 171), and optionally wherein theantibody Fc region comprises a S267E amino acid substitution, a L328Famino acid substitution, or both, and/or a N297A or N297Q amino acidsubstitution, wherein the numbering of the residues is according to EUnumbering; (c) the anti-Siglec-9 antibody has an IgG2 isotype andcomprises one or more amino acid substitutions in the Fc region at aresidue position selected from the group consisting of: P238S, V234A,G237A, H268A, H268Q, V309L, A330S, P331S, C214S, C232S, C233S, S267E,L328F, M252Y, S254T, T256E, H268E, N297A, N297Q, A330L, and anycombination thereof, wherein the numbering of the residues is accordingto EU numbering; (d) the anti-Siglec-9 antibody has a human or mouseIgG4 isotype and comprises one or more amino acid substitutions in theFc region at a residue position selected from the group consisting of:L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E,E233P, F234V, L234A/F234A, S228P, S241P, L248E, T394D, N297A, N297Q,L235E, and any combination thereof, wherein the numbering of theresidues is according to EU numbering; or (e) the anti-Siglec-9 antibodyhas a hybrid IgG2/4 isotype, and optionally wherein the antibodycomprises an amino acid sequence comprising amino acids 118 to 260 ofhuman IgG2 and amino acids 261 to 447 of human IgG4, wherein thenumbering of the residues is according to EU numbering.
 6. Theanti-Siglec-9 antibody of claim 2, wherein the anti-Siglec-9 antibody isan antibody fragment that binds to human Siglec-9 or mammalian Siglec-9protein.
 7. The anti-Siglec-9 antibody of claim 6, wherein the fragmentis an Fab, Fab′, Fab′-SH, F(ab′)2, Fv, or scFv fragment.
 8. Theanti-Siglec-9 antibody of claim 2, wherein the anti-Siglec-9 antibody isa murine antibody, a humanized antibody, a bispecific antibody, amonoclonal antibody, a multivalent antibody, a conjugated antibody, or achimeric antibody.
 9. The anti-Siglec-9 antibody of claim 2, wherein theanti-Siglec-9 antibody has dissociation constant (KD) for human Siglec-9and mammalian Siglec-9 that ranges from about 10 nM to about 10 pM,wherein the KD is determined at a temperature of approximately 25° C.10. The anti-Siglec-9 antibody of claim 2, wherein the anti-Siglec-9antibody has dissociation constant (KD) for human Siglec-9 that rangesfrom about 9 nM to about 230 pM, wherein the KD is determined at atemperature of approximately 25° C.
 11. The isolated anti-Siglec-9antibody of claim 1, wherein the anti-Siglec-9 antibody decreases cellsurface levels of Siglec-9 without inhibiting interaction betweenSiglec-9 and one or more Siglec-9 ligands.
 12. The anti-Siglec-9antibody of claim 11, wherein the antibody decreases cell surface levelsof Siglec-9 in vivo.
 13. The anti-Siglec-9 antibody of claim 11, whereinthe anti-Siglec-9 antibody inhibits one or more Siglec-9 activities. 14.The anti-Siglec-9 antibody of claim 11, wherein the one or more Siglec-9ligands are selected from the group consisting of Siglec-9 ligandsexpressed on red blood cells, Siglec-9 ligands expressed on bacterialcells, Siglec-9 ligands expressed on apoptotic cells, Siglec-9 ligandsexpressed on nerve cells, Siglec-9 ligands expressed on glia cells,Siglec-9 ligands expressed on microglia, Siglec-9 ligands expressed onastrocytes, Siglec-9 ligands expressed on tumor cells, Siglec-9 ligandsexpressed on viruses, Siglec-9 ligands expressed on dendritic cells,Siglec-9 ligands bound to beta amyloid plaques, Siglec-9 ligands boundto Tau tangles, Siglec-9 ligands on disease-causing proteins, Siglec-9ligands on disease-causing peptides, Siglec-9 ligands expressed onmacrophages, Siglec-9 ligands expressed on neutrophils, Siglec-9 ligandsexpressed on natural killer cells, Siglec-9 ligands expressed onmonocytes, Siglec-9 ligands expressed on T cells, Siglec-9 ligandsexpressed on T helper cells, Siglec-9 ligands expressed on cytotoxic Tcells, Siglec-9 ligands expressed on B cells, Siglec-9 ligands expressedon tumor-imbedded immunosuppressor dendritic cells, Siglec-9 ligandsexpressed on tumor-imbedded immunosuppressor macrophages, Siglec-9ligands expressed on myeloid-derived suppressor cells, Siglec-9 ligandsexpressed on regulatory T cells, secreted mucins, sialic acid, sialicacid-containing glycolipids, sialic acid-containing glycoproteins,alpha-2,8-disialyl containing glycolipids, branched alpha-2,6-linkedsialic acid-containing glycoproteins, terminal alpha-2,6-linked sialicacid-containing glycolipids, terminal alpha-2,3-linked sialicacid-containing glycoproteins, and di sialogangliosides.
 15. Theanti-Siglec-9 antibody of claim 11, wherein the antibody decreases cellsurface levels of Siglec-9 in vitro on primary cells selected from thegroup consisting of dendritic cells, bone marrow-derived dendriticcells, monocytes, microglia, macrophages, neutrophils, and NK cells, oron cell lines.
 16. The anti-Siglec-9 antibody of claim 11, wherein theanti-Siglec-9 antibody does not reduce TREM2 expression.
 17. Theanti-Siglec-9 antibody of claim 11, wherein the anti-Siglec-9 antibodybinds to one or more amino acids within amino acid residues 185-194 ofSEQ ID NO: 1, or amino acid residues on a mammalian Siglec-9 proteincorresponding to amino acid residues 185-194 of SEQ ID NO:
 1. 18. Theanti-Siglec-9 antibody of claim 11, wherein the anti-Siglec-9 antibodybinds to one or more amino acid residues selected from the groupconsisting of D189, P190, and R194 of SEQ ID NO: 1, or one or more aminoacid residues on a mammalian Siglec-9 protein corresponding to an aminoacid residue selected from the group consisting of D189, P190, and R194of SEQ ID NO:
 1. 19. The anti-Siglec-9 antibody of claim 11, wherein theSiglec-9 protein is a mammalian protein or a human protein.
 20. Theanti-Siglec-9 antibody of claim 11, wherein the anti-Siglec-9 antibodybinds Siglec-9 in a pH dependent manner.
 21. A pharmaceuticalcomposition comprising the anti-Siglec-9 antibody of claim 2, and apharmaceutically acceptable carrier.
 22. A humanized form of theanti-Siglec-9 antibody of claim
 1. 23. The anti-Siglec-9 antibody ofclaim 1, wherein the heavy chain variable domain comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 116-126, andthe light chain variable domain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 61-71.